Engineered immune cells that modulate receptor expression

ABSTRACT

The present disclosure provides engineered immune cells and methods for their creation and use. The immune cells comprise activating and blocking receptors, and binding of the blocking receptors to cognate ligands causes reduced surface expression of the activating receptors.

TECHNICAL FIELD

The present disclosure relates to engineered immune cells that have anenhanced safety profile and large therapeutic window.

SEQUENCE LISTING

The present application is being filed with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA2TH-001-01US-Sequence-Listing.txt, created on Sep. 20, 2021 and is 404kilobytes in size. The information in electronic format of the SequenceListing is incorporated by reference in its entirety.

BACKGROUND

Approximately 1.8 million people per year are diagnosed with a form ofcancer in the United States. Similarly, it is estimated that 23.5million Americans suffer from an autoimmune disease, almost all of whichdecrease life expectancy. Despite continual advances in treatment,education, and detection, there are over 600,000 deaths per yearattributed to cancer in the U.S., while autoimmune diseases remain aleading cause of death among patients under the age 65.

Engineered immune cells have been touted as potentially effectivetreatments for a variety of severe conditions like cancer, viralinfections, auto-immune ailments, and organ transplant rejection. Theseimmune cells, whether chimeric antigen receptor (CAR)-engineered cellsor T cell receptor (TCR)-engineered cells, often show efficaciousresults in vitro. However, in vivo, these results are rarely duplicated.Often, these treatments show a lack of efficacy in vivo and/or producesuch severe side effects, that they cannot be used as therapeutics.Thus, despite decades of consistent research, only two CAR T celltherapies have received FDA approval—Kymriah™ for acute lymphoblasticleukemia and Yescarta™ for diffuse large B-cell lymphoma.

SUMMARY OF THE INVENTION

The present disclosure provides engineered immune cells that comprisetwo types of modular, engineered ligand binding receptors caused to beexpressed on the surface of the cells. The first of these receptors isan activating receptor, which is designed to activate when bound to acognate ligand on the surface of another cell, causing it to trigger anactivating signal. The immune cells are engineered such that when thestrength of the activating signal crosses a threshold, it causes acytotoxic response by the immune cell, killing the cell expressing thecognate ligand. The second of these receptors is a blocking receptor,which when bound to a cognate blocking ligand on the surface of anon-target cell, is designed to activate and trigger a blocking signal.The blocking signal blocks the activating signal, which prevents thecytotoxic response against the non-target cell.

Generally, the activating receptors are designed to bind to cognateactivating ligands that are expressed on both target cells, such astumor cells, and non-target cells. The blocking ligands may be expressedonly by non-target cells, or expressed at lower levels by target cellscompared to non-target cells. In this way, when the engineered immunecells contact target cells, the activating receptors bind to theactivating ligands, which leads to the cytotoxic response. In contrast,when the engineered immune cells contact non-target cells, the blockingreceptors bind to the blocking ligands, blocking the cytotoxic response.This designed scheme provides the general means by which the engineeredimmune cells safely kill target cells, while limiting effects onnon-target cells. However, the immune cells of the present disclosurehave been engineered to provide several other advantageous features thatexpand their therapeutic window and efficacy, while limiting deleteriouseffects.

One of these advantageous features is that, when activated, the blockingreceptors have been designed to reduce cell surface expression of theactivating receptors. Thus, as the immune cells circulate to areas of apatient's body lacking target cells, the levels of activating receptorsexpressed on the surface of the cell are reduced. The receptors can beconfigured such that this reduction is reversible upon the activatingreceptors binding to their cognate ligands in the absence of theblocking ligands. This intentionally lowers the likelihood that acytotoxic response will be triggered in the absence of an appropriatetarget, which enhances the safety profile of the immune cells.

Further, by engineering the immune cells to reduce the expression ofactivating receptors in the absence of an appropriate target cell, theimmune cells are less likely to exhibit chronic activation and/orligand-independent tonic signaling. As a result, the immune cells of thepresent disclosure are designed to limit immune cell exhaustion,differentiation, and activation-induced immune cell death, whileconcurrently exhibiting high generation and persistence.

A further advantage of the engineered immune cells of the presentdisclosure is that, when they contact target and/or non-target cells,the activating and blocking receptors are designed to diffuse intoregions on the immune cell surface proximate to the target and/ornon-target cells. The receptors form micro-clusters in these regions. Inmicro-clusters proximate to non-target cells, the blocking receptorsbind to cognate ligands on the proximate non-target cells. The receptorscan be configured such that cross-talk between the receptors causes alocalized reduction in surface expression of the activating receptors,recruits more blocking receptors to the micro-cluster, and preventsbreakup of the micro-cluster. This leads to a localized signal thatblocks cytotoxic effects on the non-target cell.

In contrast, when the engineered immune cells contact target cells,activating receptors in micro-clusters proximate to the target cells areactivated. This leads to a localized signal that, when it passes athreshold, triggers a cytotoxic response by the immune cell that killsthe proximate target cells. The immune cells and receptors can beconfigured such that binding of the activating receptors to theircognate ligands may also locally reverse any reduced surface expressionof the activating receptor. This ensures a sufficiently strongactivating signal to trigger the cytotoxic response on the proximatetarget cell.

The immune cells of the present disclosure can form these aforementionedmicro-clusters when simultaneously contacting both target and non-targetcells. This ensures an appropriate, localized response that kills targetcells, while minimizing deleterious effects on non-target cells.

The immune cells of the present disclosure also feature blockingreceptors that are engineered to produce a ligand-dependent signal thatdominates and blocks the activating signal from the activatingreceptors. This ensures that the immune cells can be configured topossess a strong safety profile with a wide therapeutic window.

Moreover, in some methods and systems of the disclosure, the engineeredimmune cells can be produced based on the levels of blocking andactivating ligands expressed by non-target cells. Because the blockingreceptors can be tuned to have a signal that dominates initial contactwith a non-target cells, a sufficiently safe immune cell can beproduced, without relying on a large surplus of blocking receptorsexpressed as compared to activating receptors. Further, the ability ofthe blocking receptors to reduce the surface expression of theactivating receptors ensures that this level of safety increases in thepresence of non-target cells.

Another advantage conferred by the immune cells of the presentdisclosure is that receptors can be produced using modular receptorcomponents. Thus, the immune cells can be readily engineered to havereceptor pairs that target desired ligands expressed on target andnon-target cells. Moreover, the modular receptor components can be usedand interchanged to tune or adjust the relative signal strengths of eachreceptor type. This ensures that an engineered immune cell's receptorsprovide a sufficiently strong activation signal, which can be adequatelyblocked to prevent non-target effects. A surprising discovery is thatthis modular nature extends to both chimeric antigen receptors (CAR) andT cell receptors (TCR). Not only are CARs and TCRs of the presentdisclosure able to interact with each other, but parts of CARs and TCRscan be interchanged to produce customized receptors and cells.

The relative signal strength and activity of each receptor type can alsobe modulated based on cross-talk between receptors. A surprising featureof the present disclosure is, not only that cross-talk can impact signalstrength and activity, but that the impact of this cross-talk can changedepending on the distance between pairs of blocking and activatingreceptors. As the distance between a blocking receptor and activatingreceptor decreases, the impact of this cross-talk increases. Thus, thepresent disclosure provides engineered immune cells configured toexpress receptors such that they are proximate to one another to ensureoptimal interaction and strong cross-talk.

The receptors may be designed, for example, with physiochemicalproperties that ensure the receptors have a desired spacing. Thisspacing may ensure a maximum level of cross-talk between receptorsand/or ensure that the receptors do not diffuse close enough to, forinstance, invert the blocking receptor signal. The receptors can beengineered, for example, to have opposing charges or steric hindrancesto prevent them from moving too close to one another. Alternatively, orin addition, the immune cells may be engineered to have receptors thatare covalently linked to achieve a desired spacing. For example, a rigidcovalent linker between the receptors can hold the receptors at adesired spacing from one another. The rigid linker concurrently keepsthe receptors close enough to ensure cross-talk while maintain adequatespacing to prevent the blocking receptor from, for instance, invertingor becoming ligand-independent.

Another feature of the present disclosure is that the blocking receptorcan be designed using interchangeable hinges that connect anextracellular ligand binding domain to a transmembrane domain and/or anintracellular domain. The hinges can be designed to have differentlengths and flexibilities. The length and flexibility of a hinge can beused to tune the strength of the blocking signal. Longer and/or moreflexible hinges can be used to increase the strength of the blockingreceptor's signal or surface expression. In contrast, the blockingreceptor can be engineered with shorter and/or more rigid hinges todecrease the strength of the blocking receptor's signal or surfaceexpression. The blocking receptor can be configured to use a hingeselected from a group of hinges that have a known impact on the halfmaximal concentration (EC₅₀) of the activating ligand for the activatingreceptor to cause the immune cell to trigger a cytotoxic response. Thisallows pairs of blocking and activating receptors to be chosen orengineered to exhibit a desired level of activation/inhibition.

Thus, the present disclosure provides engineered immune cells, andmethods for reliably producing them, with a large therapeutic window,i.e., cells with a large range between their minimum effective dose andmaximum tolerated dose. The cells possess target-sensitive receptorsthat produce an activation signal sufficient to trigger cytotoxiceffects when encountering target cells, while concurrently producingminimal non-target effects. The engineered immune cells of the presentdisclosure also exhibit low exhaustion, differentiation, tonicsignaling, and activation-induced immune cell death, and other featuresconsistent with effective in vitro and in vivo function.

In one aspect, the present disclosure provides an engineered immune cellthat includes an activating receptor expressed on a surface of theengineered immune cell. Binding of the activating receptor to anactivating ligand on a target cell promotes a cytotoxic response by theengineered immune cell. The immune cell also includes a blockingreceptor expressed on the surface of the engineered immune cell. Bindingof the blocking receptor to a blocking ligand on a target cell causesthe engineered immune cell to exhibit reduced surface expression of theactivating receptor. High exogenous IL-2 may overcome this level ofregulation, though the activation/blockade is still enforced by otherfeatures of intracellular signaling of the activator and blockerreceptors.

Binding of the blocking receptor to the blocking ligand on the targetcell may also cause the blocking receptor to trigger an inhibitorysignal that blocks the activating signal, thereby preventing thecytotoxic response by the immune cell. The engineered immune may have aninhibitory signal dominates and blocks the activating signal.

The reduced surface expression of the activating receptor of the cellsof the present disclosure may be reversible. The reduced surfaceexpression of the activating receptor may reverse upon the engineeredimmune cell binding to the activating ligand on a target cell in theabsence of the blocking ligand. The reduced surface expression of theactivating receptor may be localized to a region of the engineeredimmune cell surface proximal to the blocking receptor. When a pluralityof the blocking receptor binds to a plurality of the blocking ligand,the reduced surface expression may be localized to regions of theengineered immune cell surface proximal to blocking receptors.

When the immune cell encounters a target cell having both the blockingand activating ligands, a plurality of activating and blocking receptorsdiffuse into a region on the of the immune cell surface proximal to thetarget cell and form a micro-cluster. In the micro-cluster, binding ofblocking receptors to the blocking ligands causes the engineered immunecell to exhibit reduced surface expression of the activating receptor inthe micro-cluster.

In certain immune cells of the disclosure, the blocking receptor cannotbind to the blocking ligand until the activating receptor binds to theactivating ligand.

The present disclosure also provides method for treating a cancer usingthe immune cells of the disclosure. In certain methods of thedisclosure, the method includes providing an engineered immune cell to apatient, wherein the engineered immune cell comprises an activatingreceptor and a blocking receptor, each expressed on a surface of theengineered immune cell. In certain methods, when the engineered immunecell encounters a tumor cell of the patient, the activating receptorbinds to an activating ligand on the tumor cell while the blockingreceptor remains unbound. This promotes a cytotoxic response by theengineered immune cell that results in a cytotoxic effect on the tumorcell. When the engineered immune cell encounters a normal cell of thepatient, the blocking receptor binds to a blocking ligand on the normalcell and causes the engineered immune cell to exhibit reduced surfaceexpression of the activating receptor. This causes a signal from theblocking receptor to dominate a signal from the activating receptor,which prevents the cytotoxic response by the engineered immune cell.

In certain methods, the reduced surface expression of the activatingreceptor is temporary. The reduced surface expression may be reversible.The reduced surface expression may be reversed upon the engineeredimmune cell binding to the first ligand on a tumor cell.

In certain methods of the disclosure, the reduced surface expression ofthe activating receptor may be localized to a region of the engineeredimmune cell surface proximal to the blocking receptor bound to theblocking ligand on the normal cell. A plurality of the blocking receptormay bind to a plurality of the blocking ligand on the normal cell, andthe reduced surface expression may be localized to the region of theengineered immune cell surface proximal to the plurality of the blockingreceptor.

A further aspect of the disclosure are methods of producing anengineered immune cell with activating and blocking receptors. Themethods of the disclosure may include, producing an engineered immunecell that expresses activating receptors and blocking receptors based ona ratio of a quantity of an activating ligand to a quantity of ablocking ligand that are expressed in non-tumor cells of a patient.

In certain methods, a tumor cell of a patient expresses the activatingligand and does not express the blocking ligand.

In certain methods of the disclosure, binding of the activatingreceptors to the activating ligands triggers an activating signal thatpromotes a cytotoxic response by the engineered immune cell.Additionally, binding of the blocking receptors to blocking ligands on anon-tumor cell may cause the blocking receptors to trigger an inhibitorysignal that blocks the activating signal.

In certain methods of the disclosure, the engineered immune cellexpresses the blocking and activating receptors at a ratio based on theratio of the quantity of the activating ligand to the quantity of theblocking ligand that are expressed in the non-tumor cells of thepatient.

In some methods, the inhibitory signal of one of the blocking receptorsdominates and blocks the activating signal of one of the activatingreceptors.

In certain methods, the ratio of the blocking receptors to theactivating receptors is less than 1. The ratio of the blocking receptorsto the activating receptors, needed to achieve a blocking signal toprovide a certain level of blocking for the activating signal, may beinversely proportional to the quantity of the activating ligandexpressed on non-tumor cells of the patient. In certain methods, whenthe immune cell contacts a non-tumor cell of the patient the blockingreceptors bind to blocking ligands on the non-tumor cell and reversiblyincrease the ratio of blocking receptors to activating receptorsexpressed by the immune cell.

In certain methods of the disclosure, each blocking receptor comprises aligand binding domain (LBD), a hinge, transmembrane domain, andintracellular domain (ICD), and the LBD, hinge, and ICD have a knowneffect on the strength of the inhibitory signal. Each activatingreceptor may comprise a ligand binding domain (LBD), a hinge,transmembrane domain, and the LBD has a known effect on the activationsignal.

The present disclosure also provides a method of producing an engineeredimmune cell that includes obtaining a sample from a patient comprisingtarget and non-target cells; performing an assay to determine a ratio ofa quantity of an activating ligand to a quantity of a blocking ligandexpressed on the non-target cells; and producing an engineered immunecell that expresses activating receptors and blocking receptors based onthe determined ratio.

In certain methods, the target cells express the activating ligand anddo not express the blocking ligand.

In some methods, binding of the activating receptors to the activatingligands triggers an activating signal that promotes a cytotoxic responseby the engineered immune cell; and binding of the blocking receptors toblocking ligands on a non-target cell causes the blocking receptors totrigger an inhibitory signal that blocks the activating signal.

In some methods, the engineered immune cell expresses the blocking andactivating receptors at a ratio based on the ratio of the quantity ofthe activating ligand to the quantity of the blocking ligand that areexpressed in the non-target cells of the patient.

In certain methods, the inhibitory signal of one of the blockingreceptors dominates and blocks the activating signal of one of theactivating receptors. The ratio of the blocking receptors to theactivating receptors is less than 1 in certain methods. The ratio of theblocking receptors to the activating receptors may be inverselyproportional to the quantity of the activating ligand expressed onnon-target cells of the patient. In some methods, when the immune cellcontacts a non-target cell of the patient the blocking receptors bind toblocking ligands on the non-target cell and reversibly increases theratio of blocking receptors to activating receptors expressed by theimmune cell.

In certain methods of the disclosure, each blocking receptor comprises aligand binding domain (LBD), a hinge, transmembrane domain, andintracellular domain (ICD), and the LBD, hinge, and ICD have a knowneffect on the strength of the inhibitory signal. Each activatingreceptor may comprise a ligand binding domain (LBD), a hinge,transmembrane domain, and the LBD has a known effect on the activationsignal.

In a further aspect, the present disclosure provides engineered immunecells with activating and blocking receptors that exhibit cross-talkbetween receptors. Thus, the present disclosure provides an engineeredimmune cell with an activating receptor that triggers a cytotoxic signalthat promotes a cytotoxic response of the engineered immune cell whenthe activating receptor binds a first ligand of a target cell; ablocking receptor that sends an interfering signal that inhibits thecytotoxic response of the engineered immune cell when the blockingreceptor binds a second ligand of the target cell, wherein cross-talkbetween the activating receptor and the blocking receptor affects anactivation threshold for the cytotoxic response.

In certain immune cells, in the absence of the first and second ligands,the effect of the cross-talk on the activation threshold is minimizedand/or reduced. The effect of the cross-talk on the activation thresholdmay increase with proximity of the activating receptor to the blockingreceptor.

In certain immune cells of the disclosure, the activating receptor andblocking receptor are covalently linked together, or havephysicochemical properties favoring interaction with one another suchthat the receptors are proximal to one another.

In some immune cells of the disclosure, when the blocking receptor bindsto the second ligand, the cross-talk between the blocking and activatingreceptors causes the immune cell to exhibit reduced surface expressionof the activating receptor.

An immune cell of the disclosure may include a plurality of theactivating and blocking receptors, and when the immune cell contacts atarget cell the plurality of the activating and blocking receptorsdiffuses into a region on the surface of the immune cell proximal to thetarget cell and forms a micro-cluster in which the effect of thecross-talk on the activation threshold is localized.

In some immune cells of the disclosure, cross-talk between theactivating receptor and the blocking receptor prevents the blockingreceptor from binding to the second ligand until the activating receptorbinds to the first ligand.

The present disclosure also provides methods for treating cancer usingthe immune cells of the present disclosure. Certain methods may includeproviding an engineered immune cell to a patient, wherein the engineeredimmune cell comprises an activating receptor and a blocking receptor,each expressed on a surface of the engineered immune cell. Theactivating receptor may trigger a cytotoxic signal that promotes acytotoxic response of the engineered immune cell when the activatingreceptor binds a first ligand of a target cell; and the blockingreceptor may send an interfering signal that inhibits the cytotoxicresponse of the engineered immune cell when the blocking receptor bindsa second ligand of the target cell, wherein cross-talk between theactivating receptor and the blocking receptor affects an activationthreshold for the cytotoxic response.

In certain methods, the absence of the second ligand, the effect of thecross-talk on the activation threshold is minimized and/or reduced. Theeffect of the cross-talk on the activation threshold may increase withproximity of the activating receptor to the blocking receptor.

In certain methods, the activating receptor and blocking receptor arelinked together or have physicochemical properties favoring interactionwith one another, such that the receptors are proximal to one another.

In certain methods, when the blocking receptor binds to the secondligand, the cross-talk between the blocking and activating receptorscauses the immune cell to exhibit reduced surface expression of theactivating receptor. The immune cell may include a plurality of theactivating and blocking receptors, and when the immune cell contacts atarget cell the plurality of the activating and blocking receptorsdiffuses into a region on the surface of the immune cell proximal to thetarget cell and forms a micro-cluster in which the effect of thecross-talk on the activation threshold is localized.

In methods of the disclosure, the cross-talk between the activatingreceptor and the blocking receptor may prevent the blocking receptorfrom binding to the second ligand until the activating receptor binds tothe first ligand.

The present disclosure also provides methods of producing engineeredimmune cells as disclosed herein. Certain methods include, determiningan amount of cross-talk between an activating receptor and a blockingreceptor for an engineered immune cell, wherein the amount of cross-talkbetween the activating receptor and the blocking receptor affects anactivation threshold for the cytotoxic response; and producing anengineered immune cell that expresses different concentrations ofactivating receptors and blocking receptors based on the determinedamount of cross-talk between the activating receptor and the blockingreceptor.

In some methods for producing immune cells, in the absence of cognateligands for the activating and blocking receptors, the amount of thecross-talk is minimized and/or reduced. The methods may includeproducing an engineered immune cell that expresses differentconcentrations of activating receptors and blocking receptors is furtherbased on a ratio of a quantity of an activating ligand to a quantity ofa blocking ligand that are expressed in non-tumor cells of a sample.

The cross-talk between the activating receptor and the blocking receptormay prevent the blocking receptor from binding to the blocking liganduntil the activating receptor binds to the activating ligand. In certainmethods, an amount of the cross-talk between the activating receptor andblocking receptor increases with proximity of the activating receptor tothe blocking receptor.

Methods include producing immune cells where the activating receptor andblocking receptor may be covalently linked, or have physicochemicalproperties favoring interaction with one another such that the receptorsare proximal to one another.

In a further aspect, the present disclosure provides engineered immunecells with activating and blocking receptors in which the blockingreceptor provides an inhibitory signal that dominates the activationsignal from the activating receptor.

Thus, the present disclosure includes an engineered immune cell with anactivating receptor on the surface of the engineered immune cell,wherein binding of the activating receptor to a first ligand on a targetcell causes the activating receptor to trigger an activating signal thatpromotes a cytotoxic response by the engineered immune cell; and ablocking receptor on the surface of the immune cell, wherein binding ofthe blocking receptor to a second ligand on a target cell causes theblocking receptor to trigger an inhibitory signal stronger than theactivating signal such that the inhibitory signal dominates and blocksthe activating signal from the activating receptor, thereby preventing alocalized cytotoxic response by the engineered immune cell.

In certain immune cells of the disclosure, binding of the blockingreceptor to the second ligand may cause the engineered immune cell toexhibit reduced surface expression of the activating receptor. Thereduced surface expression may be reversible.

The immune cells may include a plurality of activating and blockingreceptors and the ratio of the blocking receptors to the activatingreceptors expressed by the immune cells is less than or equal to 1.

In certain immune cells of the disclosure, the blocking receptor doesnot bind to the second ligand until the activating receptor binds to theactivating ligand.

In certain cells, the inhibitory signal may be localized to a region ofthe engineered immune cell surface adjacent to the blocking receptor.Similarly, the activation signal may be localized to a region of theengineered immune cell surface adjacent to the activating receptor.

When the immune cells of the disclosure encounter a target cell havingboth the first and second ligands, a plurality of activating andblocking receptors may diffuse into a region on the of the immune cellsurface proximal to the target cell and form a micro-cluster in whichthe blocking receptors prevent the localized cytotoxic response by theengineered immune cells. Binding of the blocking receptors in themicro-cluster to the second target antigen may prevent breakup of themicro-cluster. When the immune cells simultaneously contact a secondtarget cell having the first ligand and lacking the second ligand, asecond plurality of the activating receptors may diffuse into a secondregion on the surface of the immune cells proximal to the second targetcell and form a second micro-cluster that promotes the localizedcytotoxic response by the engineered immune cells that results in acytotoxic effect on the second target cell.

The present disclosure also provides methods for treating cancer usingthe immune cells of the present disclosure. The methods include a methodin which an engineered immune cell is provided to a patient, wherein theengineered immune cell comprises an activating receptor and a blockingreceptor, each expressed on a surface of the engineered immune cell,wherein: when the engineered immune cell encounters a tumor cell, theactivating receptor binds to a first ligand on the tumor cell and theactivating receptor triggers an activating signal in the engineeredimmune cell that promotes a cytotoxic response by the engineered immunecell that results in a cytotoxic effect on the tumor cell; and when theengineered immune cell encounters a normal cell, the activating receptorbinds to the first ligand on the normal cell and the blocking receptorbinds to a second ligand on the normal cell, wherein the activatingreceptor triggers an activating signal in the engineered immune cell andthe blocking receptor triggers an inhibitory signal in the engineeredimmune cell that is stronger than the activating signal such that theinhibitory signal dominates and blocks the activating signal from theactivating receptor, thereby preventing a localized cytotoxic responseby the engineered immune cell.

In some methods, binding of the blocking receptor to the second ligandcauses the engineered immune cell to exhibit reduced surface expressionof the activating receptor. The reduced surface expression may bereversible.

In methods of the disclosure, the immune cell may express differentconcentrations of the activating and blocking receptors based on a ratioof a quantity of the first ligand to a quantity of a second ligandexpressed in a normal cell of the patient. The ratio of theconcentration of blocking receptors expressed to activating receptorsexpressed may be less than or equal to 1.

In certain methods, when the immune cell encounters at least one tumorcell, a first plurality of the activating receptors diffuses into afirst region on the surface of the immune cell proximal to the tumorcell and forms a first micro-cluster that promotes the localizedcytotoxic response by the immune cell that results in a cytotoxic effecton the tumor cell. When the immune cell simultaneously encounters anormal cell, a plurality of the activating and blocking receptors maydiffuse into a second region on the surface of the immune cell proximalto the normal cell and form a second micro-cluster causing theinhibitory signal from the blocking receptors to dominate the activatingsignal from the activating receptors in the second micro-clusterpreventing the localized cytotoxic response by the engineered immunecell on the normal cell. Binding of the blocking receptors in the secondmicro-cluster to the second ligand may prevent breakup of the secondmicro-cluster.

In some methods of the disclosure, the blocking receptor does not bindto the second ligand until the activating receptor binds to theactivating ligand.

Some methods include cross-talk between the activating receptor and theblocking receptor that affects an activation threshold for the localizecytotoxic response.

In a further aspect, the present disclosure provides engineered immunecells with activating and blocking receptors that have multiplex andlocalized activity. An immune cell of the disclosure may includeactivating and blocking receptors on a surface of the cell. When theengineered immune cell encounters a tumor cell and a healthy cell afirst region of the activating and blocking receptors forms proximal tothe healthy cell and blocking receptors in the first region inhibitcytotoxic effects on the healthy cell, while, simultaneously, a secondregion of the activating and blocking receptors forms proximal to thetumor cell and promotes a cytotoxic response by the engineered immunecell that exhibits cytotoxic effects on the tumor cell.

The activating and blocking receptors in the first region may bind tocognate activating and blocking ligands on the healthy cell, and theactivating receptors in the second region may bind to cognate activatingligands on the tumor cell. The activating and blocking receptors mayform a first micro-cluster in the first region, and the activating andblocking receptors may form a second micro-cluster in the second region.

Binding of the blocking receptors in the first micro-cluster to thecognate blocking ligands on the healthy cell may cause the engineeredimmune cell to exhibit reduced surface expression of the activatingreceptor in the first micro-cluster. Binding of the blocking receptorsin the first micro-cluster to the cognate blocking ligands on thehealthy cell may prevent breakup of the first micro-cluster.

The immune cell may express different concentrations of the activatingand blocking receptors based on a ratio of a quantity of the activatingligand to a quantity of the blocking ligand expressed in a healthy cell.The ratio of the concentration of blocking receptors to activatingreceptors expressed by the immune cell may be less than or equal to 1.

In certain immune cells of the disclosure, the blocking receptors do notbind to the cognate blocking ligands until the activating receptors bindto cognate activating ligands.

In some immune cells of the disclosure, the cytotoxic response by theengineered immune cell that exhibits cytotoxic effects on the tumor cellis localized to the second region. The localized cytotoxic response doesnot exhibit cytotoxic effects on the healthy cell.

The present disclosure also provides methods for treating cancer usingthe immune cells of the disclosure. A method for treating cancer mayinclude providing an engineered immune cell to a patient, the engineeredimmune cell comprising activating and blocking cell-surface receptors.When the engineered immune cell encounters a tumor cell and a healthycell of the patient, a first set of the activating and blockingreceptors collect into a first cell-surface region of the engineeredimmune cell proximal to the healthy cell in which the blocking receptorsinhibit cytotoxic effects of the engineered immune cell on the healthycell. Simultaneously, a second set of the activating and blockingreceptors collect into a second cell-surface region of the engineeredimmune cell proximal to the tumor cell in which the activating receptorspromote a cytotoxic response by the engineered immune cell that killsthe tumor cell.

The activating and blocking receptors in the first cell-surface regionmay bind to cognate activating and blocking ligands on the healthy cell,and the activating receptors in the second cell-surface region may bindto cognate activating ligands on the tumor cell.

The activating and blocking receptors may form a first micro-cluster onthe first cell-surface region, and the activating and blocking receptorsmay form a second micro-cluster on the second cell-surface region.

Binding of the blocking receptors in the first micro-cluster to thecognate blocking ligands on the healthy cell may cause the engineeredimmune cell to exhibit reduced surface expression of the activatingreceptor in the first micro-cluster. Binding of the blocking receptorsin the first micro-cluster to the cognate blocking ligands on thehealthy cell may prevent breakup of the first micro-cluster.

In some methods of the disclosure, the immune cell expresses differentconcentrations of the activating and blocking receptors based on a ratioof a quantity of the activating ligand to a quantity of a blockingligand expressed in a healthy cell. The ratio of the concentration ofblocking receptors to activating receptors expressed by the immune cellmay be less than or equal to 1.

In certain methods, blocking receptors do not bind to the cognateblocking ligands until the activating receptors bind to the cognateactivating ligands.

In certain methods of the disclosure, the cytotoxic response islocalized to the second cell-surface region. The localized cytotoxicresponse does not exhibit cytotoxic effects on the healthy cell.

In a further aspect, the present disclosure provides immune cells thathave activating and blocking receptors that form micro-clusters on thesurface of the immune cells.

An engineered immune cell of the disclosure may include activating andblocking receptors on a surface of the engineered immune cell, wherein:when the engineered immune cell encounters a tumor cell, a firstplurality of the activating receptors diffuse into a first region on thesurface of the engineered immune cell and form a first micro-clusterproximal to the tumor cell that promotes a cytotoxic response by theengineered immune cell that results in cytotoxic effects on the tumorcell; and when the engineered immune cell encounters a normal cell, asecond plurality of the activating and blocking receptors diffuse into asecond region on the surface of the engineered immune cell and form asecond micro-cluster proximal to the normal cell, wherein the blockingreceptors in the second micro-cluster inhibit cytotoxic effects on thenormal cell.

The activating receptors in the first micro-cluster may bind to cognateactivating ligands on the tumor cell, and the activating and blockingreceptors in the second micro-cluster may bind to cognate activating andblocking ligands on the normal cell. When the immune cell encounters anormal cell, the second micro-cluster may mediate formation of acomplementary cluster of ligands on the normal cell. Binding of theblocking receptors in the second micro-cluster to the cognate blockingligands on the normal cell may cause the engineered immune cell toexhibit reduced surface expression of the activating receptor in thesecond micro-cluster. Binding of the blocking receptors in the secondmicro-cluster to the cognate blocking ligands on the normal cell mayprevent breakup of the second micro-cluster.

In some immune cells of the disclosure, when the immune cellsimultaneously contacts a normal cell and a tumor cell, the firstplurality of the activating receptors diffuse into the first region andform the first micro-cluster proximal to the tumor cell that promotesthe cytotoxic response by the engineered immune cell that results incytotoxic effects on the tumor cell; and the second plurality of theactivating and blocking receptors diffuse into the second region andform the second micro-cluster proximal to the normal cell in which theblocking receptors inhibit cytotoxic effects on the normal cell.Expression of the activating receptor in the second micro-cluster may bereduced after the second micro-cluster forms.

The immune cell may express different concentrations of the activatingand blocking receptors based on a ratio of a quantity of the activatingligand to a quantity of a blocking ligand expressed in a normal cell ofa patient. The ratio of the concentration of blocking receptorsexpressed to activating receptors expressed is less than or equal to 1.

In some immune cells of the disclosure, the blocking receptors do notbind to the cognate blocking ligands until the activating receptors bindto the cognate activating ligands.

The present disclosure provides methods for treating cancer using theimmune cells disclosed herein. A method for treating cancer may includeproviding an engineered immune cell to a patient, the engineered immunecell comprising activating and blocking cell-surface receptors.

When the engineered immune cell encounters a normal cell, a firstplurality of the activating and blocking receptors collect into amicro-cluster within a region of the cell-surface of the engineeredimmune cell proximal to the normal cell, wherein binding of one of theblocking receptors in the micro-cluster to a blocking ligand on thenormal cell inhibits breakup of the micro-cluster, and wherein theengineered immune cell kills tumor cells that exhibit an activatingligand bound by the activating receptor and do not exhibit the blockingligand such that the blocking receptor remains unbound.

When the immune cell simultaneously contacts a normal cell and a tumorcell, a first plurality of the activating receptors may diffuse into thefirst region and form the first micro-cluster proximal to the tumor cellthat promotes the cytotoxic response by the engineered immune cell thatresults in cytotoxic effects on the tumor cell, and a second pluralityof the activating and blocking receptors may diffuse into the secondregion and form the second micro-cluster proximal to the normal cell,wherein binding of one of the blocking receptors in the secondmicro-cluster to a first ligand on the normal cell inhibits breakup ofthe micro-cluster.

Binding of the blocking receptors in the second micro-cluster to thefirst ligands on the normal cell inhibits the cytotoxic effects on thenormal cell. The cytotoxic effects on the tumor cell may be localizedproximal to the first micro-cluster.

Binding of the blocking receptor to the first ligand on the normal cellmay cause a plurality of the first ligand on the normal cell to diffuseinto a region proximal to the immune cell and form a complementarymicro-cluster. Binding of the blocking receptors in the micro-cluster tothe first ligand on the normal cell may cause the engineered immune cellto exhibit reduced surface expression of the activating receptor in themicro-cluster. The reduced surface expression may be reversible.

The immune cell may express different concentrations of the activatingand blocking receptors based on a ratio of a quantity of the firstligand to a quantity of the second ligand expressed on a normal cell.The ratio of the concentration of blocking receptors to activatingreceptors expressed by the immune cell may be less than or equal to 1.

In certain methods of the disclosure, the blocking receptors in themicro-cluster do not bind to the first ligands until the activatingreceptors bind to the second ligands on the normal cell.

In a further aspect, the present disclosure provides engineered immunecells and methods of making and using them wherein the immune cellscomprise a hinge that modulates an effect of the blocking signal and/orreceptors.

Thus, the present disclosure provides a method of producing anengineered immune cell, the method including, causing an immune cell toexpress cell surface activating receptors and blocking receptors,wherein the blocking receptors comprise a selected hinge. Binding of theactivating receptors to activating ligands on a target cell triggers anactivating signal that promotes a cytotoxic response by the immune cell.Binding of the blocking receptors to blocking ligands on a non-targetcell causes the blocking receptors to trigger a blocking signal thatinhibits the activating signal. The selected hinge is selected tomodulate an effect of the blocking signal on the activating signal.

In certain methods, the selected hinge comprises a peptide having acertain length, and the length of the peptide modulates the effect ofthe blocking signal on the activating signal. The effect of the blockingsignal on the activating signal may be an increased inhibition of theactivating signal. The increased inhibition of the activating signal mayincrease a half maximal effective concentration (EC₅₀) of the activatingligand for the activating receptors to promote the cytotoxic response.

In certain methods and immune cells of the disclosure, cross-talk and/orstructure function interactions between the hinge of the blockingreceptor and the activating receptor further impart different signalstrengths for the blocking receptor.

The effect of the blocking signal on the activating signal may be adecreased inhibition of the activating signal, and the length of thepeptide is less than about 24 amino acids.

In certain aspects of the disclosure, the engineered immune cell iscaused to express the blocking and activating receptors at a ratio, andthe ratio blocking receptors to activating receptors expressed isdecreased as the length of the peptide of the selected hinge isincreased. The peptide may further have a degree of flexibility, and thepeptide's length and degree of flexibility modulate the effect of theblocking signal on the activating signal.

The selected hinge may be selected from hinges that each have a knowneffect on the EC₅₀ of the activating ligand for the activating receptorsto promote the cytotoxic response. The length of the peptide of eachhinge that may have a known effect on the EC₅₀ of the activating ligandis between 10 and 64 amino acids in length. The peptide of the selectedhinge may be at least 24 amino acids in length. The peptide of theselected hinge may be at least 64 amino acids in length. In certainaspects, the hinge having a peptide of 64 amino acids in length causesat least a fifty-fold increase in the EC₅₀ relative to a hinge having apeptide of 10 amino acid in length. In certain aspects, the hinges thateach have a known effect on the EC50 comprise hinges 2B1, 2B1 truncated,PD-1, CTLA4, BTLA, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQID NO: 395, SEQ ID NO: 396 and SEQ ID NO: 397.

In certain aspects of the disclosure, the length of the peptideincreases surface expression of the blocking receptor.

In certain aspects, the hinge peptide is derived from leukocyteimmunoglobulin-like receptor subfamily B member 1 (LILRB1).

In certain aspects of the disclosure, the hinge comprises a peptidehaving a degree of flexibility, and the peptide's degree of flexibilitymodulates the effect of the blocking signal on the activating signal.The peptide may be a flexible peptide and the effect of the blockingsignal on the activating signal is an increased inhibition of theactivating signal. The flexible peptide may comprise glycine-glutaminerepeats and/or glycine-serine repeats.

In certain aspects, the hinge comprises a rigid peptide, and the peptideand the effect of the blocking signal on the activating signal isreduced inhibition of the activating signal. The rigid peptide mayinclude, for example, an alpha-helix, repeats of (XP) where X is anyamino acid, and/or repeats consisting of alanine, glutamic acid, andlysine.

In certain aspects, the present disclosure provides engineered immunecells, and methods of making and using the same, wherein activating andblocking receptors are spaced apart by at least an average minimumdistance on the immune cell surface.

Thus, in certain aspects, the disclosure provides a method of producingan engineered immune cell, the method including, causing an immune cellto express cell surface activating and blocking receptors. Binding ofthe activating receptor to an activating ligand on a target celltriggers an activating signal that promotes a cytotoxic response by theengineered immune cell. Binding of the blocking receptor to a blockingligand on a non-target cell causes the blocking receptor to trigger ablocking signal that inhibits the activating signal. Wherein thereceptors remain spaced apart by at least an average minimum distance onthe immune cell surface.

The blocking signal of a blocking receptor may invert to an activatingsignal when a blocking receptor is spaced at a distance less than theaverage minimum distance from the activating receptor.

In certain aspects, the method also includes determining the distanceless than the average minimum distance at which the blocking signalinverts.

In certain methods, the average minimum distance is about 100-1000angstroms. In certain methods, the average minimum distance is aboutbetween about 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 angstroms. Incertain methods, the average minimum distance is greater or equal to 200angstroms. In certain methods, the average minimum distance is about 200angstroms.

In certain methods, each receptor has a ligand binding domain (LBD), ahinge, a transmembrane domain, and an intracellular domain (ICD).

Certain methods further include covalently or non-covalently linking thereceptors via a spacer such that the receptors are separated by a knownspacing. The spacer may comprise a C- or N-terminal fusion. Thereceptors may be linked to the spacer via the LBD or ICD of eachreceptor. The receptors may be linked to the spacer at their respectivehinge. The spacer may comprise one or more moieties that allownon-covalent binding of the receptors at their respective hinge. Thespacer may comprise, for example, two moieties, that are independentlyfused to the LBD, ICD, or hinge of each receptor. The receptors may belinked via a spacer that comprises a non-covalent interacting motif thatmediates protein-protein interaction, such as leucine zipper. Thereceptors may be covalently attached via the spacer, and the spacer maycomprise a cleavable linker such as a disulfide linker.

In certain aspects, the receptors are linked via a spacer that comprisesa rigid peptide linker. The rigid peptide may include, for example, analpha-helix, repeats of XP where X is any amino acid, and/or repeatsconsisting of alanine, glutamic acid, and lysine.

In certain aspects, the receptors have physiochemical properties thatprevent the receptors from being spaced at a distance less than theaverage minimum distance. The physiochemical properties may include, forexample, opposite charges engineered by design on the receptorsequences, leading to attraction, compared to neutral or similarcharges. The physiochemical properties may also or alternativelyinclude, for example, steric effects, non-covalent interactions, and/orvan der Waals interactions.

In certain aspects, the present disclosure includes an engineered immunecell comprising a cell surface activating receptor and a cell surfaceblocking receptor, wherein each of the cell surface activating receptorand the cell surface blocking receptor comprise physiochemicalproperties that prevent the cell surface activating receptor and thecell surface blocking receptor from being spaced at a distance less thanan average minimum distance.

In certain aspects, the average minimum distance is about 100-1000angstroms. In certain methods, the average minimum distance is about1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 angstroms. Incertain methods, the average minimum distance is greater or equal to 200angstroms. In certain methods, the average minimum distance is about 200angstroms.

In certain aspects, the receptors have physiochemical properties thatprevent the receptors from being spaced at a distance less than theaverage minimum distance. The physiochemical properties may include, forexample, opposite charges engineered by design on the receptorsequences, leading to attraction, compared to neutral or similarcharges. The physiochemical properties may also or alternativelyinclude, for example, steric effects, non-covalent interactions, and/orvan der Waals interactions.

In a further aspect, the present disclosure provides an engineeredimmune cell comprising a cell surface activating receptor, a cellsurface blocking receptor, and a spacer operably associated with thecell surface activating receptor and the cell surface blocking receptor,wherein the spacer is configured to maintain the cell surface activatingreceptor and the cell surface blocking receptor spaced apart by at leastan average minimum distance on the immune cell surface.

In certain aspects, the average minimum distance is about 100-1000angstroms. In certain methods, the average minimum distance is about1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 angstroms. Incertain methods, the average minimum distance is greater or equal to 200angstroms. In certain methods, the average minimum distance is about 200angstroms.

In certain aspects, each receptor of the engineered immune cell has aligand binding domain (LBD), a hinge, a transmembrane domain, and anintracellular domain (ICD).

The spacer may covalently or non-covalently link the receptors such thatthe receptors are separated by a known spacing. The spacer may comprisea C- or N-terminal fusion. The receptors may be linked to the spacer viathe LBD or ICD of each receptor. The receptors may be linked to thespacer at their respective hinge. The spacer may comprise one or moremoieties that allow non-covalent binding of the receptors at theirrespective hinge. The spacer may comprise, for example, two moietiesthat are independently fused to the LBD, ICD, or hinge of each receptor.The receptors may be linked via a spacer that comprises a non-covalentinteracting motif that mediates protein-protein interaction, such asleucine zipper. The receptors may be covalently attached via the spacer,and the spacer may comprise a cleavable linker such as a disulfidelinker.

In certain aspects, the receptors are linked via a spacer that comprisesa rigid peptide linker. The rigid peptide may include, for example, analpha-helix, repeats of XP where X is any amino acid, and/or repeatsconsisting of alanine, glutamic acid, and lysine.

In a further aspect, the disclosure provides a method for treatingcancer that includes providing an engineered immune cell to a patient,wherein the engineered immune cell comprises an activating receptor anda blocking receptor, each expressed on a surface of the engineeredimmune cell. The activating receptor triggers a cytotoxic signal thatpromotes a cytotoxic response of the engineered immune cell when theactivating receptor binds a first ligand of a target cell. The blockingreceptor sends an interfering signal that inhibits the cytotoxicresponse of the engineered immune cell when the blocking receptor bindsa second ligand of the target cell. The receptors remain spaced apart byat least an average minimum distance on the immune cell surface.

In certain methods, the average minimum distance is about 100-1000angstroms. In certain methods, the average minimum distance is about1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 angstroms. Incertain methods, the average minimum distance is greater or equal to 200angstroms. In certain methods, the average minimum distance is about 200angstroms.

In certain methods, the receptors possess physiochemical properties thatprevent the cell surface activating receptor and the cell surfaceblocking receptor from being spaced at a distance less than an averageminimum distance.

In certain aspects, the receptors have physiochemical properties thatprevent the receptors from being spaced at a distance less than theaverage minimum distance. The physiochemical properties may include, forexample, opposite charges engineered by design on the receptorsequences, leading to attraction, compared to neutral or similarcharges. The physiochemical properties may also or alternativelyinclude, for example, steric effects, non-covalent interactions, and/orvan der Waals interactions.

In certain aspects, the immune cell includes a spacer operablyassociated with the cell surface activating receptor and the cellsurface blocking receptor, wherein the spacer is configured to maintainthe cell surface activating receptor and the cell surface blockingreceptor spaced apart by at least an average minimum distance on theimmune cell surface.

In certain methods, each receptor has a ligand binding domain (LBD), ahinge, a transmembrane domain, and an intracellular domain (ICD).

The spacer may covalently or non-covalently link the receptors such thatthe receptors are separated by a known spacing. The spacer may comprisea C- or N-terminal fusion. The receptors may be linked to the spacer viathe LBD or ICD of each receptor. The receptors may be linked to thespacer at their respective hinge. The spacer may comprise one or moremoieties that allow non-covalent binding of the receptors at theirrespective hinge. The spacer may comprise, for example, two moietiesthat are independently fused to the LBD, ICD, or hinge of each receptor.The receptors may be linked via a spacer that comprises a non-covalentinteracting motif that mediates protein-protein interaction, such asleucine zipper. The receptors may be covalently attached via the spacer,and the spacer may comprise a cleavable linker such as a disulfidelinker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the immune cells expressing activating andblocking receptors.

FIGS. 2-3 show reduced surface expression of activating receptors andreversibility of reduced surface expression of activating receptors.

FIGS. 4-6 provide experimental results showing reduced expression ofactivating receptors.

FIG. 7 provides experimental results showing reduced expression ofactivating receptors.

FIG. 8 shows a schematic for an experiment to show the reversibility ofreduced expression of activating receptors.

FIGS. 9-12 provide experimental results showing reversibly reducedexpression of activating receptors.

FIGS. 13-16 provide experimental results showing reversibly reducedexpression of activating receptors.

FIGS. 17-19 shows experimental results indicating that the blockingreceptor does not undergo reduced surface expression in an appreciableamount in the presence of non-target cells.

FIG. 20 shows a schematic of micro-clustering.

FIG. 21 shows a schematic of multiplex and localized signaling by theactivating and blocking receptors.

FIG. 22 provides experimental results showing that activity of theblocking receptors is ligand-dependent.

FIG. 23 provides experimental results showing that the blockingreceptors cause minimum ligand-independent inhibition of the activatingreceptors.

FIG. 24 provides a schematic of the activating and blocking receptors.

FIG. 25 provides experimental results for the effect the hinge of theblocking receptor has on blocking strength.

FIG. 26 provides experimental results showing that the identity of theligand binding domain of the activating receptor drives the activity ofthe receptor.

FIG. 27 provides experimental results showing the effect the ligandbinding domain has on the blocking receptor.

FIG. 28 shows combinations of various CAR- and TCR-based activating andblocking receptors.

FIG. 29 provides experimental results showing that a CAR-based blockingreceptor can inhibit a TCR-based activating receptor.

FIG. 30 provides experimental results showing that a CAR ligand bindingdomain can be used with a TCR activating receptor intracellular domain.

FIG. 31 shows different receptors that can be created in accordance withthe present disclosure.

FIG. 32 provides experimental results showing the effect theintracellular domain has on the strength of the blocking receptorsignal.

FIGS. 33-34 provide experimental results that indicate cross-talkbetween receptors.

FIG. 35 shows various ways to control the distance between activatingand blocking receptors.

DETAILED DESCRIPTION

The present disclosure provides engineered immune cells featuring “ANDNOT” Boolean logic by expressing engineered activating and blockingreceptors. The cells are designed such that when the activatingreceptors bind to cognate activating ligands on a target cell, theyproduce an activating signal. If the strength of the activating signalcrosses a threshold, it causes a cytotoxic response by the immune cell,killing the cell expressing the cognate ligands. The second of thesereceptors is a blocking receptor, which is designed to bind to a cognateblocking ligand on the surface of another cell, thereby activating thereceptor and causing it to trigger a blocking signal that blocks theactivating signal, which prevents the cytotoxic response. The “AND NOT”Boolean logic engineered into the immune cells of the present disclosuremakes them ideal for use as therapeutic agents.

Thus, in an exemplary method of the disclosure, a patient diagnosed witha medical condition, such as cancer, is treated with engineered immunecells that target and kill the patient's cancer cells while preservingtheir normal, healthy cells. One or more cellular samples may be takenfrom the patient, such as from a blood draw or tumor biopsy. Targetcells, such as tumor cells, are identified in the sample. The identifiedtarget cells are assayed to determine the levels of expression of one ormore cell-surface ligands. This may include, for example, assessing RNAexpression profiles for various cell-surface receptors or using antibodyprobes that bind to certain cell surface receptors. Assaying targetcells may determine, for example, that target cells do not express acertain cell surface ligand due to a loss of heterozygosity.

Then, immune cells, such as T cells, are harvested from the patient.These cells are caused to express engineered activating and blockingreceptors. The blocking receptor is designed to bind to a blockingligand expressed on healthy cells of the patient. This blocking ligandmay be chosen because it is lost from cancer cells, e.g., due to loss ofheterozygosity. The activating receptor is designed to bind to anactivating ligand that is expressed on both healthy cells and cancercells of the patient.

After the engineered immune cells are proliferated, the cells areadministered to the patient. The immune cells are designed such thatwhen an immune cell encounters a cancer cell in the patient's body, theactivating receptors bind to activating ligands on the cancer cell. Thistriggers a cytotoxic immune response by the immune cell that kills thecancer cell. When the immune cell encounters a healthy cell theactivating and blocking receptors bind to activating and blockingligands on the healthy cell. Binding of the blocking receptors toblocking ligands inhibits and blocks the cytotoxic immune responsetriggered by the activating receptors binding to activating ligands. Inthis way, the engineered immune cells are designed to limit deleteriouseffects on non-target cells.

FIG. 1 shows a schematic of this “AND NOT” Boolean logic in the immunecells of the present disclosure. In FIG. 1, the immune cells 103comprise a blocking receptor 105 and an activating receptor 107. Anon-target cell 109 expresses an activating ligand 111 and blockingligand 113. When the immune cell 103 contacts the non-target cell, theactivating receptor 107 binds to the activating ligand 111 triggering anactivating signal 117. Concurrently, the blocking receptor 105 binds tothe blocking ligand 113, triggering a blocking signal 115. As shownschematically, when the immune cell 103 contacts a non-target cell, thestrength of blocking signal is greater than the activating signal 119.Binding both blocking ligands and activating ligands causes the “ANDNOT” state in the immune cell. The signal from the blocking receptorsblocks the activating signal. As such, the activating signal cannot passthe threshold to trigger a cytotoxic response, which prevents adeleterious effect on the non-target cell expressing both ligands.

Conversely, a target cell 121, such as a tumor cell, expresses ablocking ligand 111, but does not express an activating ligand, orexpresses an activating ligand at a lower level compared to thenon-target cell 109. Thus, when the immune cell 103 contacts the targetcell 121, the activating receptor 107 binds to the activating ligand111, triggering an activating signal 117. As shown schematically, whenthe immune cell 103 contacts a non-target cell, the strength of theactivating signal is greater than the blocking signal 123. When thestrength of the activating signal crosses an activation threshold, theimmune cell produces a cytotoxic response 125 that kills the target cell121.

Generally, the cognate antigens chosen for the activating receptors areexpressed on both target cells, such as tumor cells, and non-targetcells. The selected blocking ligands are expressed only by non-targetcells, or expressed at lower levels by target cells compared tonon-target cells. In this way, when the engineered immune cells contacttarget cells, the activating receptors bind to the activating ligands,which leads to the cytotoxic response. In contrast, when the engineeredimmune cells contact non-target cells, the blocking and activatingreceptors bind to their cognate blocking and activating ligands. Thiscompletes the “AND NOT” Boolean logic, thereby blocking the cytotoxicresponse. This scheme provides the general means by which the engineeredimmune cells safely kill target cells while limiting effects onnon-target cells.

Engineered immune cells have been used as cancer therapies, such asimmunotherapies. Traditionally, engineered immune cells have beendesigned to target molecular targets such as neo-antigens. Neo-antigensare a class of somatic mutant proteins that are mutated during somaticgrowth of tumors. They provide ideal targets for immune cell therapiesbecause they comprise variants not found on non-target, healthy cells ofa patient. However, very few cancers express neo-antigens. Thus,different targets must be pursued to treat most types of cancer usingengineered immune cells. However, in prior immune cell therapies thatlacked the blocking receptors of the present disclosure, when the immunecells targeted antigens expressed by healthy and non-health cells,severe adverse effects arose due to non-target activity. In cancerimmunology, this phenomenon is known as on-target, off-tumorrecognition.

The engineered immune cells and receptors of the present disclosureprovide greater flexibility in choice of molecular target. The efficacyof the blocking receptor ensures that non-target effects are limited.Thus, the immune cells of the present disclosure can be designed totarget widely expressed, cell surface molecules as the activatingligand. Exemplary ligands include a cell adhesion molecule, a cell-cellsignaling molecule, an extracellular domain, a molecule involved inchemotaxis, a glycoprotein, a G protein-coupled receptor, atransmembrane, a receptor for a neurotransmitter or a voltage gated ionchannel.

Activating receptors of the present disclosure may be configured totarget activating ligands that are encoded by genes with essentialcellular functions. Advantageously, this can prevent antigen escape,increasing the long-term efficacy of the engineered immune cells as atherapeutic. By selecting activating ligands encoded by genes withessential cellular functions, loss or escape of the ligand, such asthrough aneuploidy in cancer cells, is less likely. Thus, the activatingligand may be encoded by a gene that is haploinsufficient, i.e., loss ofcopies of the gene encoding the ligand are not tolerated by the cell andlead to cell death, or a disadvantageous mutant phenotype. In fact, theengineered immune cells of the present disclosure may be designed totarget activating ligands expressed on all cells of patient.

Advantageously, because the immune cells of the present disclosure canbe engineered to use widely-expressed activating ligands, severalproblems can be avoided. For example, prior engineered immune cellsoften targeted minimally expressed antigens, such as certainneo-antigens. Thus, to ensure an adequate activating signal, priorimmune cells were engineered with activating receptors that had veryhigh expression or affinities for their activating ligands.

However, merely increasing the density or affinity of receptors isinadequate to ensure efficacy. High receptor affinity can lead toproportionally severe, toxic effects on non-target cells. It can alsohinder an engineered immune cell from disassociating from a target cell,which limits the ability of the immune cell to subsequently bind to andkill other target cells. Further, high affinity can cause receptors tobe continually activated. This chronic activation can lead to immunecell exhaustion, reduced generation and persistence, increasingdifferentiation to undesired phenotypes, and activation-induced immunecell death. Increasing density can lead to similar effects throughligand-independent, tonic signaling.

Use of widely expressed activating ligands, made safe through the use ofa blocking receptor, allows the engineered immune cells of the presentdisclosure to avoid these potential issues.

The blocking receptor can be designed to bind to a cell surface moleculenot expressed on the surface of the target cell, or expressed atsufficiently low levels on a target cell. Thus, where the engineeredimmune cells are used to treat cancer, the blocking ligand may be chosenbased on the loss of heterozygosity (LOH) of the target cancer cells,i.e., the cancer cells no longer express the ligand due to a loss ofgenetic material from one of the homologous chromosomes. Exemplary geneswhose expression is frequently lost in cancer cells, for example due toLOH, include, HLA class I alleles, minor histocompatibility antigens(MiHAs), and Y chromosome genes (in males where the homologouschromosome is the X chromosome).

As will be discussed, the immune cells of the present disclosure possessseveral features that leverage the general nature of the “AND NOT”Boolean logic, to provide effective, target-specific effects whileminimizing deleterious non-target effects.

Reduced Activator Expression

Surprisingly, the engineered immune cells of the present disclosure,which express activating and blocking receptors, can be designed toexhibit reduced surface expression of activating receptors when theycontact non-target cells.

This ability to reduce surface expression is shown schematically in FIG.2. When an engineered immune cell contacts non-target cells, theblocking receptors and activating receptors bind to their cognateactivating ligands and blocking ligands expressed on the non-targetcell. As explained, this causes the blocking signal to inhibit theactivating signal. Further, activation of the blocking receptors causesreduced surface expression of the activating receptors. The activatingreceptors may be internalized, such that they are no longer on thesurface of the immune cell and able to interact with activating ligands.As a result, the threshold to trigger a cytotoxic response by the immunecell is raised. Thus, the immune cells may temporarily exhibit a reducedpropensity to kill cells, which can increase the therapeutic window ofthe cells.

As shown in FIG. 3, when the immune cell contacts a target cell, thisreduced surface expression of the activating receptor does not occurand/or is reversed. Thus, when the immune cell encounters a target cell,the activating receptors bind to activating ligands. This provides theactivating signal, but also causes the immune cell to reverse thereduced surface expression of the activating receptors. When theactivating receptors are expressed on the surface of the immune cell athigher numbers, the activation threshold to trigger the cytotoxicresponse is reduced. Thus, immune cells in contact with target cells maytemporarily exhibit an increased propensity to kill cells.

In certain immune cells of the disclosure, the reduced and/or regainedexpression of the activating receptor can be localized to a region ofthe immune cell surface proximate to a target or non-target cell. Thus,returning to FIG. 3, when the immune cell contacts a non-target cells,reduction of the activating receptors occurs in regions 303 proximate tothe non-target cells. This can desensitize the immune cell in theregions 303 proximate to each non-target cell, which raises theactivation threshold to trigger the cytotoxic response. Similarly, theimmune cell can contact a target cell, and reduced surface expression ofthe activating receptor is reversed and/or does not occur in a region(s)305 proximate to the target cell(s). This allows the region 305proximate to the target cell to experience a local activation signalsufficient to trigger a localized cytotoxic response.

Advantageously, these localized responses can occur as an immune cellsimultaneously and/or sequentially contacts target and non-target cells.Thus, as shown in FIG. 3, the immune cells can provide an activatingsignal localized to a region 305 proximate to a target cell, while alsomodulating expression of the activating receptor to reduce theactivation threshold in the same region. Simultaneously, the immune cellcan provide localized blocking signals and localized, reduced expressionof the blocking receptor.

Reducing surface expression of the activating receptors. when not incontact with target cells. confers several advantages to the immunecells of the present disclosure. For example, if an immune cellcirculates away from target cells, such as in a tumor, the immune cellis presumably more likely to contact non-target cells. By reducing thesurface expression of the activating receptor in response to a lack oftarget cells, the immune cell increases its activation threshold, whichcan temporarily reduce the propensity of the immune cell to trigger acytotoxic response. This designed feature of the engineered immune cellsacts as a “safe mode”, which enhances the safety and protective effectsprovided by the “AND NOT” Boolean logic, and further limits deleteriouseffects caused by the immune cells.

Further, while in this “safe mode”, fewer activating receptors areavailable to activate. Thus, the immune cells of the present disclosureare less likely to experience chronic activation or ligand-independenttonic signaling. As a result, the immune cells are less susceptible toexhaustion, differentiation, and activation-induced immune cell death,while concurrently exhibiting high generation and persistence.

An additional and important feature of this reduced expression is thatit does not extend to the engineered blocking receptors. Only theengineered activating receptors experience appreciable amounts ofreduced expression. This ensures the safety profile of the immune cellsof the present disclosure is maintained.

Thus, the present disclosure provides an engineered immune cell with anactivating receptor and blocking receptor expressed on a surface of theengineered immune cell, wherein binding of the activating receptor to anactivating ligand on a target cell promotes a cytotoxic response by theengineered immune cell, and binding of the blocking receptor to ablocking ligand causes the engineered immune cell to exhibit reducedsurface expression of the activating receptor.

The present disclosure also provides a method for treating a cancer thatincludes providing an engineered immune cell to a patient, wherein theengineered immune cell comprises an activating receptor and a blockingreceptor, each expressed on a surface of the engineered immune cell.When the engineered immune cell encounters a tumor cell of the patient,the activating receptor binds to an activating ligand on the tumor cellwhile the blocking receptor remains unbound, thereby promoting acytotoxic response by the engineered immune cell that results in acytotoxic effect on the tumor cell. When the engineered immune cellencounters a normal cell of the patient the blocking receptor binds to ablocking ligand on the normal cell and causes the engineered immune cellto exhibit reduced surface expression of the activating receptor,thereby causing a signal from the blocking receptor to dominate a signalfrom the activating receptor and prevent the cytotoxic response by theengineered immune cell.

Micro-Clusters

A further advantageous feature of the engineered immune cells disclosedherein is that the cells and receptors can be designed such that theactivating and blocking receptors form micro-clusters on the surface ofthe immune cell. This ability to form micro-clusters provides anengineered immune cell with the ability to sense its proximity totarget/non-target cells and provide an appropriate, localized response.

FIG. 20 shows a schematic of the micro-clustering behavior. When animmune cell encounters a target or non-target cell, itsactivating/blocking receptors bind to cognate ligands on the encounteredcell(s). Cross-talk between these bound receptors and unbound receptorson the surface of the immune cell causes the unbound receptors todiffuse to a region on the immune cell surface proximate to theencountered cell(s). When the receptors diffuse into this region, theyform a micro-cluster in which the ligand binding domains of thereceptors locate in an activation synapse between the immune cell andthe encountered cell(s).

Forming a micro-cluster with both activating and blocking receptorsensures that, when the blocking receptors are activated in the presenceof an appropriate ligand on a non-target cell, the blocking signal istriggered proximate to the activation signal. This ensures that theblocking receptors can provide a localized inhibitory effect on theactivation signal, thereby protecting the non-target cell. Thus, themicro-clusters enhance the “AND NOT” Boolean logic conferred by theactivating and blocking receptors.

Advantageously, the engineered immune cells can be configured such thatthe activating and/or blocking ligands on target and/or non-target cellsto experience a similar clustering effect on the surface of theencountered cell(s). Unbound activating and blocking ligands diffuseinto an area on the target/non-target cell surface proximate to theimmune cell, and become available for binding to a cognate receptor inthe activation synapse.

Receptors are held in place on the surface of the immune cell by bindingto cognate ligands in the activation synapse. This ensures that thereceptors remain confined to a micro-cluster while the immune cell is incontact with a target/non-target cell. Maintaining the receptors withina micro-cluster helps assure that adequate numbers of activating and/orblocking receptors are within a region proximate to an encounteredcell(s) to provide the requisite activating or blocking signal. It alsoincreases the relative strength of both the activating and blockingreceptors, which widens the therapeutic window of the immune cells ofthe disclosure.

The ability of the receptors to form micro-clusters also enhances thelocalized, reduced expression of the activating receptors when an immunecell encounters a non-target cell. By bringing activating and blockingreceptors in close proximity, e.g., within the confines of amicro-cluster, the effect of cross-talk between the receptors isincreased. This cross-talk leads to localized, reduced expression of theactivating receptors in the micro-cluster.

Thus, the present disclosure provides an engineered immune cellcomprising activating and blocking receptors on a surface of theengineered immune cell. When the engineered immune cell encounters atumor cell, a first plurality of the activating receptors diffuse into afirst region on the surface of the engineered immune cell and form afirst micro-cluster proximal to the tumor cell that promotes a cytotoxicresponse by the engineered immune cell that results in cytotoxic effectson the tumor cell. When the engineered immune cell encounters a normalcell, a second plurality of the activating and blocking receptorsdiffuse into a second region on the surface of the engineered immunecell and form a second micro-cluster proximal to the normal cell,wherein the blocking receptors in the second micro-cluster inhibitcytotoxic effects on the normal cell.

The present disclosure also includes a method for treating cancer, themethod comprising providing an engineered immune cell to a patient, theengineered immune cell comprising activating and blocking cell-surfacereceptors. When the engineered immune cell encounters a normal cell, afirst plurality of the activating and blocking receptors collect into amicro-cluster within a region of the cell-surface of the engineeredimmune cell proximal to the normal cell. Binding of one of the blockingreceptors in the micro-cluster to a blocking ligand on the normal cellinhibits breakup of the micro-cluster. The engineered immune cell killstumor cells that exhibit an activating ligand bound by the activatingreceptor and do not exhibit the blocking ligand such that the blockingreceptor remains unbound.

Multiplex and Localized Signaling

The engineered immune cells of the present disclosure have been designedto exhibit multiplex and localized activity. A shown in FIG. 21 anengineered immune cell 103 can simultaneously contact both target cells121 and non-target cells 109. On regions of the immune cell 103 surfaceproximate to a non-target cell, the activating receptors 107 andblocking receptors 105 bind to activating ligands 111 and blockingligands 113 on the non-target cell. As a result, a localized blockingsignal inhibits a cytotoxic response 125 by the immune cell on theproximate non-target cell. Simultaneously or sequentially, the immunecell 103 can contact a target cell 121. The activating receptor 107binds to the activating ligand 111 on the target cell 121. This causes alocalized cytotoxic response 125, which may release cytotoxic granules2103. The cytotoxic response only targets the target cell 121. Thelocalized inhibition of the cytotoxic response in areas proximate to thenon-target cells 109 protects them from an undesired immune response.

Thus, the present disclosure provides an engineered immune cell thatincludes activating and blocking receptors on the surface of the cell.When the engineered immune cell encounters a tumor cell and a healthycell, a first region of the activating and blocking receptors formproximal to the healthy cell and blocking receptors in the first regioninhibit cytotoxic effects on the healthy cell. Simultaneously, a secondregion of the activating and blocking receptors form proximal to thetumor cell and promotes a cytotoxic response by the engineered immunecell that exhibits cytotoxic effects on the tumor cell.

The present disclosure also provides a method for treating cancer thatincludes providing an engineered immune cell to a patient. Theengineered immune cell has activating and blocking cell-surfacereceptors. When the engineered immune cell encounters a tumor cell and ahealthy cell of the patient, a first set of the activating and blockingreceptors collect into a first cell-surface region of the engineeredimmune cell proximal to the healthy cell in which the blocking receptorsinhibit cytotoxic effects of the engineered immune cell on the healthy.Simultaneously, a second set of the activating and blocking receptorscollect into a second cell-surface region of the engineered immune cellproximal to the tumor cell in which the activating receptors promote acytotoxic response by the engineered immune cell that kills the tumorcell.

Dominant Blocking Receptors

The engineered immune cells of the present disclosure can be configuredto have blocking receptors that produce a blocking signal that canoverwhelm and fully inhibit the activating signal from the activatingreceptors.

As shown in FIG. 22, which is explained in greater detail below, thecells can be designed to express activating and blocking receptors that,when expressed at equivalent concentrations, it takes less blockingantigen relative activating antigen to inhibit the activating signal.Thus, each blocking receptor can inhibit the activating signal of one ormore activating receptors. This means that the blocking signal from asingle blocking receptor can dominate and inhibit the activating signalfrom a single activating receptor. This helps solidify the safetyprofile of the “AND NOT” Boolean logic used by the immune cells of thepresent disclosure.

As shown in FIG. 23, the blocking receptors can be engineered to provideminimal ligand-independent blocking activity on the activatingreceptors. As a corollary, the blocking receptors can provideoverwhelmingly ligand-dependent activity.

Thus, the immune cells of the present disclosure may include blockingreceptors that provide, for example, a less than 10× shift in the EC₅₀of the activating receptors when the immune cells are contacted with theactivating ligand in the absence of the blocking ligand. The immunecells of the present disclosure can provide a less than 3× shift in theEC₅₀ of the activating receptors when the immune cells are contactedwith the activating ligand in the absence of the blocking ligand.

Since the blocking receptors of the present disclosure can provide anoverwhelmingly ligand-dependent, dominate blocking signal, the levels ofactivating ligand and blocking ligand expressed on a non-target cell canbe used to inform the appropriate levels of activating and blockingreceptor expressed by the engineered immune cells of the presentdisclosure. The dominate blocking signal provides assurance that ligandquantity can be used as a proxy to inform the levels of activating andblocking receptors that should be expressed in order to assuresufficient inhibition. Moreover, the ligand-dependent nature of theblocking signal means that the expression of the blocking receptor willrequire little to no adjustment to prevent unintended increases to theEC₅₀ of the activating receptors in the absence of the blocking ligand.

Thus, the present disclosure provides methods for producing engineeredimmune cells that express activating and blocking receptors based on aratio of a quantity of activating ligands to a quantity of blockingligands that are expressed in a normal, non-tumor cell of a patient. Theactivating and blocking receptors may be expressed at a ratio based uponthe ratio of the quantity of activating ligands to the quantity ofblocking ligands expressed by the normal cell.

The present disclosure also provides an engineered immune cell with anactivating receptor on a surface of the engineered immune cell. Bindingof the activating receptor to an activating ligand on a target cellcauses the activating receptor to trigger an activating signal thatpromotes a cytotoxic response by the engineered immune cell. The cellalso has a blocking receptor. Binding of the blocking receptor to ablocking ligand on a target cell causes the blocking receptor to triggeran inhibitory signal stronger than the activating signal such that theinhibitory signal dominates and blocks the activating signal from theactivating receptor, thereby preventing a localized cytotoxic responseby the engineered immune cell.

The disclosure further includes a method for treating cancer, the methodcomprising providing an engineered immune cell to a patient. Theengineered immune cell comprises an activating receptor and a blockingreceptor, each expressed on a surface of the engineered immune cell.When the engineered immune cell encounters a tumor cell, the activatingreceptor binds to an activating ligand on the tumor cell and theactivating receptor triggers an activating signal in the engineeredimmune cell that promotes a cytotoxic response by the engineered immunecell that results in a cytotoxic effect on the tumor cell. When theengineered immune cell encounters a normal cell, the activating receptorbinds to the activating ligand on the normal cell and the blockingreceptor binds to a blocking ligand on the normal cell. This leads tothe activating receptor triggering an activating signal in theengineered immune cell and the blocking receptor triggering aninhibitory signal in the engineered immune cell that is stronger thanthe activating signal, such that the inhibitory signal dominates andblocks the activating signal from the activating receptor, therebypreventing a localized cytotoxic response by the engineered immune cell.

Modulating Activating and Blocking Signals of Receptors

The present disclosure also provides strategies for engineeringreceptors in a manner that modulates receptor signal strength to ensurestrong activation signals and sufficient blocking signals.

FIG. 24 shows a schematic of the blocking and activating receptors ofthe present disclosure. In general, each type of receptor can comprisefour parts, the ligand binding domain (“LBD”), the hinge (“H”), thetransmembrane domain (“TM”), and the intracellular domain (“ICD”). Eachof these four parts can have an impact on the structure-activityrelationship of each receptor. By altering these parts, the behavior ofeach receptor can be finely tuned to exhibit desired activity. Forexample, altering these parts can cause the receptors to exhibit varyingspecificity and affinity for cognate ligands, strengths of activatingand/or blocking signals, levels of cross-talk between receptors, and/orreceptor surface expression.

The hinge is an extracellular domain between a receptor's extracellularligand binding domain and transmembrane domain and/or intracellulardomain. Surprisingly, the Inventors of the present disclosure have foundthat, for the activating receptor, a wide variety of hinge lengths andsequences are tolerated. Thus, changes to the activating receptor hingecan provide relatively little change to the structure activityrelationship of the activating receptor. For example, changes to thehinge were shown to cause only minimal contributions to the activatingreceptors' EC₅₀, baseline signaling, and maximum signaling.

In contrast, the Inventors of the present disclosure have found thatmodifications to the hinge can be used to modulate the activity of theblocking receptor, including increases in the surface expression of theblocking receptor and blocking signal strength. Thus, a feature of thepresent disclosure is that the blocking receptor can be designed usinginterchangeable hinges that connect an extracellular ligand bindingdomain to a transmembrane domain and/or an intracellular domain.

The hinges can be designed to have different lengths and flexibilities.As shown in FIG. 24, flexible hinges inure blocking receptors with agreater blocking strength compared to rigid hinges. However, a greaterchange to blocking strength can be provided by changing the length ofthe hinge. As shown in FIG. 25, lengthening a hinge from about 25 aminoacids to about 35 amino acids confers a significant increase in blockerstrength. This increase becomes more dramatic, as the hinge lengthapproaches 65 amino acids in length. As also shown in FIG. 25, therelative flexibility/rigidity of a hinge also impacts the strength of ablocker. Although, this impact is reduced compared to that provided bythe hinge length.

Thus, the blocking receptors can be designed with longer and/or moreflexible hinges to increase the strength of the blocking receptor'ssignal or surface expression. In contrast, the blocking receptor can beengineered with shorter and/or more rigid hinges to decrease thestrength of the blocking receptor's signal or surface expression. Theblocking receptor can be configured to use a hinge selected from a groupof hinges that have a known impact on the EC₅₀ of the activating ligandfor the activating receptor to cause the immune cell to trigger acytotoxic response. This allows pairs of blocking and activatingreceptors to be chosen or engineered to exhibit a desired level ofactivation/inhibition.

Advantageously, as the activating receptor can tolerate a wide varietyof hinges, the activating receptors can be engineered with hinges thatinteract with a blocking receptor at the structural level. Differentactivator hinges may provide varying levels of interaction with aspecific blocker. Thus, various activator hinges can be tested with aparticular blocking receptor to determine the identity of activatingreceptor hinges that lead to increased blocking by a particular blockingreceptor. Such testing may include, for example, changing the hinge ofan activating receptor and measuring the blocking receptor strength,i.e., the IC₅₀, of a particular blocking receptor when a particularactivating receptor hinge is used.

The Inventors of the present disclosure found that the identity of theligand binding domain of the engineered activating receptors has thegreatest impact on the structure activity relationship of the receptors.As shown in FIG. 26, different ligand binding domains, which all bind tothe same activating ligand, provide effects on the receptors' EC₅₀ thatdiffer by orders of magnitude. In contrast, the identity of the hingeand/or intracellular domain provides a smaller impact on the receptors'EC₅₀.

As with the LBD of the activating receptor, the identity of the blockingreceptor LBD can have large effects on the IC₅₀ of the engineered immunecells of the present disclosure. This is shown in FIG. 27, where severaldifferent ligand binding domains were tested for their effect on theIC₅₀ of engineered immune cells. Interestingly, the Inventors of thepresent disclosure found that when a ligand binding domain was switchedbetween an activating receptor and blocking receptor, the LBD provided acorrelative effect on the IC₅₀ and EC₅₀ of an immune cell.

The Inventors of the present disclosure found that a wide variety ofcommonly used intracellular domains have relatively minimal impacts onthe EC₅₀ of the activating receptor. Conversely, the Inventorsdiscovered that the intracellular domain of the blocking receptor canhave large effects on the strength of the blocking signal. Thus, theintracellular domain of the blocking receptor can be changed to modulatethe strength of the blocking signal to ensure adequate inhibition. Asshown in FIG. 32, changing the intracellular domain of the blockingreceptor can have wide ranging effects on the strength of the blockingsignal.

Receptor Cross-Talk

The present disclosure also provides engineered immune cells in whichthe activity of the activating and blocking receptors is modulated viacross-talk between the receptors.

FIGS. 33-34 show the impact receptor cross-talk can have on the abilityof the blocking receptor to inhibit the activation signal. Engineeredimmune cells were created with one of five different activatingreceptors. Though the activating receptors differed between the celllines, each targeted the same activating ligand, epidermal growth factorreceptor (EGFR), using a different antibody. As shown by the five graphsat the bottom in FIGS. 33-34, each of the different activating receptorsprovided the immune cells with equivalent abilities to kill targetcells. Then, immune cells were created that had one of the fiveactivating receptors and the same blocking receptor. Addition of theblocker caused some of the immune cells, like CT486, to exhibit asignificant decrease in the cells' ability to kill target cells. Theblocking receptors also provided varying effects in the ability of theimmune cells to inhibit the activating signal in the presence ofnon-target cells.

This disparity in behavior between different activating receptors and ablocking receptor can be attributed to cross-talk between the receptors.

Thus, the present disclosure provides an engineered immune cell thatincludes an activating receptor that triggers a cytotoxic signal thatpromotes a cytotoxic response of the engineered immune cell when theactivating receptor binds to an activating ligand of a target cell, ablocking receptor that sends an interfering signal that inhibits thecytotoxic response of the engineered immune cell when the blockingreceptor binds a blocking ligand, and cross-talk between the activatingreceptor and the blocking receptor that affects an activation thresholdfor the cytotoxic response.

The disclosure also includes a method for treating cancer, the methodincludes providing an engineered immune cell to a patient. Theengineered immune cell comprises an activating receptor and a blockingreceptor, each expressed on a surface of the engineered immune cell. Theactivating receptor triggers a cytotoxic signal that promotes acytotoxic response of the engineered immune cell when the activatingreceptor binds to an activating ligand of a target cell. The blockingreceptor sends an interfering signal that inhibits the cytotoxicresponse of the engineered immune cell when the blocking receptor bindsa blocking ligand. Cross-talk between the activating receptor and theblocking receptor affects an activation threshold for the cytotoxicresponse.

Modulating Receptor Proximity

The Inventors of the present disclosure made the surprising discoverythat the strength of the blocking signal can increase as the distancebetween activating and blocking receptors decreases, and that when thereceptors are separated by a particular average minimum distance, theblocking signal provides a maximum inhibitory effect on an activatingreceptor. Thus, the present disclosure provides engineered immune cells,and methods of making using engineered immune cells, with activating andblocking receptors spaced apart by at least a minimum average distanceon the immune cell surface. The present Inventors also discovered thatwhen activating and blocking receptors are within a certain, closeproximity to one another, the activation of the blocking receptor maycause the blocking receptor to invert and provide an activating signal.

In certain engineered immune cells of the present disclosure, thisaverage minimum distance is between about 100 to 200, 200 to 300, 300 to400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900to 1000 angstroms.

As shown in FIG. 35, the Inventors devised several strategies to ensurethat the activating and blocking receptors are spaced at a distance toensure a high blocking signal strength. For example, the receptors canbe attached via a C-terminal or N-terminal bridge. Alternatively or inaddition, the receptors can be designed to have substituent groups oramino acids with opposing charges to enforce spacing between receptors.Bulky substituent groups or amino acids can also be used to cause stericeffects that prevent the receptors from diffusing too close to oneanother.

Thus, the present disclosure provides engineered immune cells withactivating and blocking receptors that possess physiochemical propertiesthat maintain an average minimum distance between the receptors on thecell surface. Physiochemical properties may include, for example,opposing charges on each of the cell surface activating receptor and thecell surface blocking receptor, non-covalent interactions, van der Wallsinteractions, and/or steric effects.

The present disclosure also or alternatively provides engineered immunecells that have a spacer operably associated with an activating andblocking receptor on the cell surface that is configured to maintain anaverage minimum distance between the receptors on the cell surface. Thespacer may covalently or non-covalently link the activating and blockingreceptors. The spacer may include C- or N-terminal fusion that links thereceptors. The spacer may alternatively or in addition include twomoieties that allow non-covalent binding between the LBD, ICD, and/orhinge of each receptor. The spacer may also or alternatively include anon-covalent interacting motif that mediates protein-proteininteraction, such as a leucine zipper.

The distance between the activating and blocking receptors may becontrolled by using a spacer that includes a linker. Any linker may beused, and many fusion protein linker formats are known. For example, thelinker may be flexible or rigid. Non-limiting examples of rigid andflexible linkers are provided in Chen et al. (Adv Drug Deliv Rev. 2013;65(10):1357-1369).

Non-limiting exemplary rigid linkers include alpha helix-forming linkerswith the sequence of (EAAAK)_(n) and (EAAAK)_(n)A, wherein n=1-10.Another exemplary rigid linker is a proline rich linker having thesequence (XP)_(n) where X is any amino acid and is preferably selectedfrom A, G, and E and n=1-10, and glycine-serine linkers with a highratio of serine to glycine.

The ligand binding domains described herein may be linked to each otherin a random or specified order. The ligand binding domains describedherein may be linked to each other in any orientation of N to Cterminus.

Optionally, a short oligo- or polypeptide linker, for example, between 2and 40 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) inlength may form the linkage between the domains. The linker is a peptideof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 amino acidresidues.

Non-limiting examples of amino acids found in linkers include Gly, Ser,Glu, Gin, Ala, Leu, Iso, Lys, Arg, Pro, and the like.

The linker may be [(Gly)n1Ser]n2, where n1 and n2 may be any number(e.g. n1 and n2 may independently be 1, 2, 4, 5, 6, 7, 8, 9, 10 or morethan 10). The linker may be flexible polypeptide linker that is aGly/Ser linker and comprises the amino acid sequence (Gly-Gly-Ser),(Gly-Gly-Gly-Ser), or (Gly-Gly-Gly-Gly-Ser) which can be repeated ntimes, where n is a positive integer equal to or greater than 1. Forexample, n−1, n−2, n−3, n−4, n−5, n−6, n−7, n−8, n=9 and n=10. Thelinker may include multiple repeats of (Gly Gly Ser), (Gly Ser) or (GlyGly Gly Ser). Also included within the scope of the invention arelinkers described in WO2012/138475 (incorporated herein by reference).In some embodiments, the flexible polypeptide linkers include, but arenot limited to, GGS, GGGGS (SEQ ID NO: 226), GGGGS GGGGS (SEQ ID NO:227), GGGGS GGGGS GGGGS (SEQ ID NO: 228), GGGGS GGGGS GGGGS GG (SEQ IDNO: 229) or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 230). In someembodiments, the linkers include multiple repeats of (Gly Gly Ser), (GlySer) or (Gly Gly Gly Ser (SEQ ID NO: 231)).

The linker sequence may comprise a long linker (LL) sequence. The longlinker sequence may comprise GGGGS, repeated four times. Such a linkermay be used to link intracellular domains in a TCR alpha fusion proteinof the disclosure. The long linker sequence may comprise GGGGS, repeatedthree times. The linker sequence may comprise a short linker (SL)sequence. The short linker sequence may comprise GGGGS. A glycine-serinedoublet can be used as a suitable linker. Alternatively, domains arefused directly to each other via peptide bonds without use of a linker.

By reducing the G:S ratio in a Gly-Ser linker, the linker can be mademore rigid.

The strength of the blocking signal may be the strongest when theactivating and blocking receptors are separated by a distance of 0-1000angstroms. The strength of the blocking signal may be the strongest whenthe activating and blocking receptors are separated by a distance of0-50 angstroms, 50-100 angstroms, 100-200 angstroms, 200-300 angstroms,300-400 angstroms, 400-500 angstroms, 500-600 angstroms, 600-700angstroms, 700-800 angstroms, 800-900 angstroms, or 900-1000 angstroms.Preferably, the distance is about 200 angstroms.

Thus, the present disclosure provides an engineered immune cell with anactivating receptor on the cell surface that triggers a cytotoxic signalthat promotes a cytotoxic response of the engineered immune cell whenthe activating receptors binds to a first ligand on a target cell; and ablocking receptor on the cell surface that sends an interfering signalthat inhibits the cytotoxic response of the engineered immune cell whenthe blocking receptor binds a second ligand of the target cell.Proximity of the blocking receptor to the activating receptor effects anactivation threshold for the cytotoxic response, and the activating andblocking receptors physiochemical properties favoring interaction withone another, such that the receptors are spaced apart at an averagedistance on the immune cell surface.

The present disclosure also provides a method for treating cancer thatincludes providing an engineered immune cell to a patient, wherein theengineered immune cell comprises an activating receptor and a blockingreceptor, each expressed on a surface of the engineered immune cell. Theactivating receptor triggers a cytotoxic signal that promotes acytotoxic response of the engineered immune cell when the activatingreceptor binds a first ligand of a target cell, and the blockingreceptor sends an interfering signal that inhibits the cytotoxicresponse of the engineered immune cell when the blocking receptor bindsa second ligand of the target cell. Proximity of the blocking receptorto the activating receptor affects an activation threshold for thecytotoxic response, and the activating and blocking receptorsphysiochemical properties favoring interaction with one another, suchthat the receptors are spaced apart at an average distance on the immunecell surface.

The present disclosure also provides a method of producing an engineeredimmune cell that includes producing an engineered immune cell thatexpresses activating receptors and blocking receptors based on adetermined distance between the receptors, wherein an activationthreshold for a cytotoxic response by the immune cell is maximized whenthe receptors are separated on the cell surface by the determinedaverage distance.

Receptor Types

The present disclosure provides immune cells comprising activating andblocking receptors, which may independently comprise a chimeric antigenreceptor (CAR) a T cell receptor (TCR) or a combination of componentsfrom CARs or TCRs.

As shown in FIG. 28, the immune cells of the present disclosure can usereceptors that comprise various combinations of TCRs and CARs. Forexample, as shown in FIG. 29, both a blocking CAR and blocking TCR caneffectively inhibit the activation signal of a TCR-based activatingreceptor.

Moreover, the receptors of the present disclosure can effectively usecomponents of both CARs and TCRs to achieve desired receptor activity.

As shown in FIG. 30, the ligand binding domain of a CAR activatingreceptor can be used with the intracellular domain of a TCR activatingreceptor, and still provide a target-specific activation signal.

As shown in FIG. 31, the various components of TCRs and CARs can beinterchanged to provide receptors with activities beyond blocking andactivating receptors. For example, the components can be used to createInverter TCRs, Super TCRs, Parasitic TCRs, and Molecular Integrators.

In some embodiments, one or more of the blocking receptor and activatingreceptor comprise a CAR. All CAR architectures are envisaged as withinthe scope of the instant disclosure.

The CARs of the present disclosure comprise an extracellular hingeregion. Incorporation of a hinge region can affect cytokine productionfrom CAR-T cells and improve expansion of CAR-T cells in vivo. Exemplaryhinges can be isolated or derived from IgD and CD8 domains, for exampleIgG1, CD8α, or CD28, such as those disclosed by the Inventors of thepresent disclosure in PCT International Application No.PCT/US2020/045250 and PCT/US2021/030149, which are incorporated hereinby reference in their entirety.

For example, exemplary hinges used in the receptors disclosed herein,which are isolated or derived from CDSa or CD28 include a CDSa hingecomprising an amino acid sequence having at least 80% identity, at least90% identity, at least 95% identity, at least 99% identity or isidentical to a sequence of SEQ ID NOS: 1 or 3 or encoded by SEQ ID NO:4.

The CARs of the present disclosure can be designed to comprise atransmembrane domain that is fused to the hinge of the CAR. Thetransmembrane domain may be naturally associated with one of the domainsof the CAR, such as the hinge or intracellular domain. For example, aCAR comprising a CD28 co-stimulatory domain might also use a CD28transmembrane domain. In some instances, the transmembrane domain can beselected or modified by amino acid substitution to avoid binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins to minimize interactions with other members of thereceptor complex.

The transmembrane domain may be derived either from a natural orsynthetic source. When the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsmay be isolated or derived from (i.e., comprise at least thetransmembrane region(s) of) the alpha, beta, or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulinsuch as IgG4.

Alternatively, the transmembrane domain may be synthetic, in which caseit can comprise predominantly hydrophobic residues such as leucine andvaline. Certain transmembrane domains may comprise a triplet ofphenylalanine, tryptophan and valine found at each end of a synthetictransmembrane domain. Optionally, a short oligo- or polypeptide linker,preferably between 2 and 10 amino acids in length may form the linkagebetween the transmembrane domain and the cytoplasmic signaling domain ofthe CAR. A glycine-serine doublet provides a particularly suitablelinker. The CARs may comprise a CD28 transmembrane domain or anIL-2Rbeta transmembrane domain, such as those disclosed by the presentInventors in PCT International Application No. PCT/US2020/045250 andPCT/US2021/030149, incorporated herein by reference.

For example, the CD28 transmembrane domain may comprise an amino acidsequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 5. The CD28 transmembrane domain may be encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to SEQ ID NO: 6. Anexemplary IL-2R beta transmembrane domain as disclosed here may comprisean amino acid sequence having at least 80% identity, at least 90%identity, at least 95% identity, at least 99% identity or is identicalto SEQ ID NO: 7. In some aspects, an exemplary IL-2Rbeta transmembranedomain is encoded by a nucleotide sequence having at least 80% identity,at least 90% identity, at least 95% identity, at least 99% identity oris identical to a sequence of SEQ ID NO: 8.

The intracellular signaling domains of CARs used as parts of theactivating or blocking receptors are responsible for activation of atleast one of the normal effector functions of the immune cell in whichthe CAR has been placed. The term “effector function” refers to aspecialized function of a cell. Effector functions of a regulatory Tcell, for example, include the suppression or downregulation ofinduction or proliferation of effector T cells. Thus, the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function.

While usually an entire intracellular signaling domain can be employed,in many cases it is not necessary to use the entire domain. To theextent that a truncated portion of the intracellular signaling domain isused, such truncated portion may be used in place of the intact chain aslong as it transduces the effector function signal. In some cases,multiple intracellular domains can be combined to achieve the desiredfunctions of CAR-T cells of the instant disclosure. The termintracellular signaling domain is thus meant to include any truncatedportion of one or more intracellular signaling domains sufficient totransduce the effector function signal.

Examples of intracellular signaling domains for use in the CARs of theinstant disclosure include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability. In certain receptors of thedisclosure, the intracellular activation domain ensures that there is Tcell receptor (TCR) signaling necessary to activate the effectorfunctions of the CAR-T cell.

The CAR intracellular domains of the instant disclosure may comprise atleast one cytoplasmic activation domain. The at least one cytoplasmicactivation domain can be a CD247 molecule (CD3) activation domain, astimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activationdomain, or a DNAX-activating protein of 12 kDa (DAP12) activationdomain, such as those disclosed by the present inventors in PCTInternational Application No. PCT/US2020/045250 and PCT/US2021/030149,which are incorporated by reference.

For example, the CD3z activation domain comprises an amino acid sequencehaving at least 80% identity, at least 90% identity, at least 95%identity, at least 99% identity or is identical to SEQ ID NO: 9 and/orencoded by a nucleotide sequence having at least 80% identity, at least90% identity, at least 95% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 10.

It is known that signals generated through a TCR alone can beinsufficient for full activation of a T cell, and that a secondary orco-stimulatory signal may be also required. Thus, T cell activation canbe mediated by two distinct classes of cytoplasmic signaling sequence:those that initiate antigen-dependent primary activation through the TCR(primary cytoplasmic signaling sequences) and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory or inhibitory manner. Exemplarycytoplasmic signaling sequences are disclosed by the present Inventorsin PCT International Application No. PCT/US2020/045250, which isincorporated by reference.

Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. In certain receptors of thedisclosure, the cytoplasmic signaling domain contains 1, 2, 3, 4, or 5ITAMs.

In certain receptors of the disclosure, the cytoplasmic domain comprisesa CD3 activation domain. The CD3ζ activation domain may comprise asingle ITAM, two ITAMs, or three ITAMs.

Further examples of ITAM containing primary cytoplasmic signalingsequences that can be used in the CARs of the instant disclosure includethose derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the instant invention comprises acytoplasmic signaling sequence derived from CD3ζ.

In certain receptors of the disclosure, the cytoplasmic domain of theCAR may comprise the CD3ζ signaling domain by itself or combined withany other desired cytoplasmic domain(s). For example, the cytoplasmicdomain of the CAR can comprise a CD3ζ chain portion and a co-stimulatorydomain.

For example, the CD3z activation domain may comprise a single ITAM andcomprises an amino acid sequence having at least 80% identity, at least90% identity, at least 95% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 11 and/or encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to SEQ ID NO: 12.

The co-stimulatory domain refers to a portion of a CAR comprising theintracellular domain of a costimulatory molecule. A costimulatorymolecule is a cell surface molecule, other than an antigen receptor orits ligands, that is required for an efficient response of lymphocytesto an antigen. In receptors of the disclosure, the costimulatory domainis selected from the group consisting of IL2Rβ, Fc Receptor gamma(FcRγ), Fc Receptor beta (FcRβ), CD3g molecule gamma (CD3γ), CD3δ, CD3ε,CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79bmolecule (CD79b), carcinoembryonic antigen related cell adhesionmolecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNFreceptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4(OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40),programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS),lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2),CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer celllectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatorydomains, or functional fragments thereof.

The cytoplasmic domains within the cytoplasmic signaling portion of theCARs of the instant disclosure may be linked to each other in a randomor specified order. Optionally, a short oligo- or polypeptide linker,for example between 2 and 10 amino acids in length may form the linkage.A glycine-serine doublet provides an example of a suitable linker.

The intracellular domains of CARs of the instant disclosure may includeat least one co-stimulatory domain. The co-stimulatory domain may beisolated or derived from CD28.

An exemplary CD28 co-stimulatory domain comprises an amino acid sequencehaving at least 80% identity, at least 90% identity, at least 95%identity, at least 99% identity or is identical to a sequence of SEQ IDNO: 13 and/or encoded by a nucleotide sequence having at least 80%identity, at least 90% identity, at least 95% identity, at least 99%identity or is identical to a sequence of SEQ ID NO: 14.

The intracellular domain of the CARs of the instant disclosure mayinclude an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta)cytoplasmic domain. The IL-2Rbeta domain may be truncated. The IL-2Rbetacytoplasmic domain may comprise one or more STAT5-recruitment motifs,which may be outside the IL-2Rbeta cytoplasmic domain.

An exemplary IL-2Rbeta intracellular domain may comprise an amino acidsequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 15 and/or encoded by a nucleotide sequence having at least 80%identity, at least 90% identity, at least 95% identity, at least 99%identity or is identical to a sequence of SEQ ID NO: 16.

Exemplary STAT5-recruitment motifs are provided by Passerini et al.,(2008) STAT5-signaling cytokines regulate the expression of FOXP3 inCD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells,International Immunology, Vol. 20, No. 3, pp. 421-431, and by Kagoya etal., (2018) A novel chimeric antigen receptor containing a JAK-STATsignaling domain mediates superior antitumor effects. Nature Medicinedoi:10.1038/nm.4478, which are each incorporated herein by reference.

An exemplary STAT-recruitment motif used herein may consist of SEQ IDNO: 17.

In certain blocking receptors of the disclosure, the inhibitory signalis transmitted through the intracellular domain of the receptor. Thus,the blocking receptor may comprise an inhibitory intracellular domain.

The inhibitory intracellular domain may comprise an immunoreceptortyrosine-based inhibitory motif (ITIM). The inhibitory intracellulardomain comprising an ITIM can be isolated or derived from an immunecheckpoint inhibitor such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immuneinhibitory receptors expressed on the surface of T cells, and play apivotal role in attenuating or terminating T cell responses.

“ITIM” refers to a conserved sequence of amino acids with a consensussequence provided in SEQ ID NO: 274. Exemplary ITIMs include, thosehaving sequences of SEQ ID NOS: 67, 68, 69, and 70. In some embodiments,the intracellular domain comprises a sequence at least 95% identical toSEQ ID NOS: 71, 72, 73, 74, 75, or 76.

Inhibitory domains can also be isolated from human tumor necrosis factorrelated apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.

The inhibitory domain may comprise an intracellular domain, atransmembrane or a combination thereof. Alternatively, the inhibitorydomain comprises an intracellular domain, a transmembrane domain, ahinge region or a combination thereof. The inhibitory domain may containan immunoreceptor tyrosine-based inhibitory motif (ITIM). The inhibitorydomain comprising an ITIM can be isolated or derived from an immunecheckpoint inhibitor such as CTLA-4 and PD-1.

Inhibitory domains can be isolated from human tumor necrosis factorrelated apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.The inhibitory domain may be isolated or derived from a human protein,for example a human TRAIL receptor, CTLA-4, or PD-1 protein. In someembodiments, the TRAIL receptor comprises TR10A, TR10B or TR10D.

Endogenous TRAIL is expressed as a 281-amino acid type II trans-membraneprotein, which is anchored to the plasma membrane and presented on thecell surface. TRAIL is expressed by natural killer cells, which,following the establishment of cell-cell contacts, can induceTRAIL-dependent apoptosis in target cells. Physiologically, theTRAIL-signaling system was shown to be essential for immunesurveillance, for shaping the immune system through regulating T-helpercell 1 versus T-helper cell 2 as well as “helpless” CD8+ T-cell numbers,and for the suppression of spontaneous tumor formation.

The inhibitory domain may comprise an intracellular domain isolated orderived from a CD200 receptor. The cell surface glycoprotein CD200receptor 1 (Uniprot ref: Q8TD46) represents another example of aninhibitory intracellular domain of the present invention. Thisinhibitory receptor for the CD200/OX2 cell surface glycoprotein limitsinflammation by inhibiting the expression of proinflammatory moleculesincluding TNF-alpha, interferons, and inducible nitric oxide synthase(iNOS) in response to selected stimuli.

The inhibitory domain may be isolated or derived from killer cellimmunoglobulin like receptor, three Ig domains and long cytoplasmic tail2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domainsand long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin likereceptor B1 (LIR1), programmed cell death 1 (PD1), Fc gamma receptor IIB(FcgRIIB), killer cell lectin like receptor K1 (NKG2D), CTLA-4, a domaincontaining a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one orboth of the N and C terminal SH2 domains), or ZAP70 KI_K369A (kinaseinactive ZAP70).

The inhibitory domain may be isolated or derived from a human protein.The blocking receptor may comprise a cytoplasmic domain andtransmembrane domain isolated or derived from the same protein. Forexample, an ITIM containing protein. The blocking receptor may comprisea cytoplasmic domain, a transmembrane domain, and an extracellulardomain or a portion thereof isolated or derived isolated or derived fromthe same protein. The blocking receptor may comprise a hinge regionisolated or derived from isolated or derived from the same protein asthe intracellular domain and/or transmembrane domain.

In certain immune cells of the disclosure, one or more of the activatingand blocking receptors comprise a T Cell Receptor (TCR).

A “TCR”, sometimes also called a “TCR complex” or “TCR/CD3 complex”refers to a protein complex comprising a TCR alpha chain, a TCR betachain, and one or more of the invariant CD3 chains (zeta, gamma, deltaand epsilon), sometimes referred to as subunits.

The TCR alpha and beta chains can be disulfide-linked to function as aheterodimer to bind to peptide-MHC complexes. Once the TCR alpha/betaheterodimer engages peptide-MHC, conformational changes in the TCRcomplex in the associated invariant CD3 subunits are induced, whichleads to their phosphorylation and association with downstream proteins,thereby transducing a primary stimulatory signal. In an exemplary TCRcomplex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma fora heterodimer, and two CD3 zeta form a homodimer.

The LBD of the activating or blocking receptors may be fused to anextracellular domain of a TCR subunit. The TCR subunit can be TCR alpha,TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. Both the first and secondligand binding domains may be fused to the same TCR subunit in differentTCR receptors. Alternatively, the first and second ligand bindingdomains are fused to different TCR subunits in different TCR receptors.

The LBD of the activating receptor and blocking receptor may eachindependently comprise an scFv domain or a Vβ-only domain.

TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gammaand CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3gamma, CD3 delta or CD3 epsilon, or fragments or derivatives thereof,can be fused to one or more domains capable of providing a stimulatorysignal of the disclosure, thereby enhancing TCR function and activity.Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta orCD3 epsilon, or fragments or derivative thereof, can be fused to aninhibitory intracellular domain of the disclosure.

The receptors of the present disclosure may comprise TCRs comprising aTCR variable domain. The TCR variable domain specifically binds to anantigen in the absence of a second TCR variable domain (a Vβ-onlydomain).

The TCRs may comprise additional elements besides the TCR variabledomain, including additional amino acid sequences, additional proteindomains (covalently associated, non-covalently associated or covalentlyand non-covalently associated with the TCR variable domain), fusion ornon-covalent association of the TCR variable domain with other types ofmacromolecules (for example polynucleotides, polysaccharides, lipids, ora combination thereof), fusion or non-covalent association of the TCRvariable domain with one or more small molecules, compounds, or ligands,or a combination thereof. Any additional element, as described, may becombined provided that the TCR variable domain is configured tospecifically bind the epitope in the absence of a second TCR variabledomain.

TCRs comprising a Vβ-only domain as described herein may comprise asingle TCR chain (e.g. α, β, γ, or δ chain), or may comprise a singleTCR variable domain (e.g. of α, β, γ, or δ chain). If a TCR is a singleTCR chain, then the TCR chain comprises a transmembrane domain, aconstant (or C domain) and a variable (or V domain), but does notcomprise a second TCR variable domain. The TCRs may comprise or consistof a TCR α chain, a TCR β chain, a TCR γ chain or a TCR δ chain. TheTCRs may be a membrane bound proteins. The TCRs may alternatively bemembrane associated proteins.

The TCRs may use a surrogate α chain that lacks a Vα segment, whichforms activation competent TCRs complexed with the six CD3 subunits. TheTCRs may function independently of a surrogate α chain that lacks a Vαsegment. For example, one or more TCRs may be fused to transmembrane(e.g., CD3ζ and CD28) and intracellular domain proteins (e.g., CD3ζ,CD28, and/or 4-1BB) that are capable of activating T cells in responseto antigen.

TCRs may comprise one or more single TCR chains fused to the Vβ-onlydomain described herein. For example, the TCRs may comprise, or consistessentially of single a TCR chain, a single β TCR chain, a single γ TCRchain, or a single δ TCR chain fused to one or more Vβ-only domains.

The TCRs may engage antigens using complementarity determining regions(CDRs). Each TCR may contain three complement determining regions (CDR1,CDR2, and CDR3).

The first and/or second ligand binding Vβ-only domain may be a human TCRvariable domain. Alternatively, the first and/or second Vβ-only domainmay be a non-human TCR variable domain. The first and/or second Vβ-onlydomain may be a mammalian TCR variable domain. The first and/or secondVβ-only domain may be a vertebrate TCR variable domain.

Where Vβ-only domain is incorporated into a fusion protein, for examplea fusion protein comprising a TCR subunit, and optionally, an additionalstimulatory intracellular domain, the fusion protein may comprise aVβ-only domain and any other protein domain or domains.

TCR receptors comprising transmembrane domains isolated or derived fromany source are envisaged as within the scope of the fusion proteins ofthe disclosure.

The TCR transmembrane domain may be one that is associated with one ofthe other domains of the fusion protein, or isolated or derived from thesame protein as one of the other domains of the fusion protein. Thetransmembrane domain and the second intracellular domain may be from thesame protein, for example a TCR complex subunit such as TCR alpha, TCRbeta, CD3 delta, CD3 epsilon or CD3 gamma. The extracellular domain(svd-TCR), the transmembrane domain and the second intracellular domainmay be from the same protein, for example a TCR complex subunit such asTCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma.

The TCR extracellular domain (comprising one or more ligand bindingdomains, such as Vβ-only domain and scFv domains), the transmembranedomain and the intracellular domain(s) may be from different proteins.For example, the engineered svd-TCR may comprise a CD28 transmembranedomain with a CD28, 4-1BB and CD3ζ intracellular domain.

The TCR transmembrane domain may be derived from a natural orrecombinant source. When the source is natural, the domain may bederived from any membrane-bound or transmembrane protein.

The transmembrane domain may be capable of signaling to theintracellular domain(s) whenever the TCR complex is bound to a target. Atransmembrane domain of particular use in this receptors of thedisclosure may include at least the transmembrane region(s) of thealpha, beta, or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154.

The transmembrane domain can be attached to the extracellular region ofthe fusion protein, e.g., the antigen binding domain of the TCR alpha orbeta chain, via a hinge, e.g., a hinge from a human protein. Forexample, in one embodiment, the hinge can be a human immunoglobulin (Ig)hinge, e.g., an IgG4 hinge, or a CD8a hinge. The hinge may be isolatedor derived from CD8α or CD28.

For example, an exemplary hinge isolated or derived from CD8a hingecomprises an amino acid sequence having at least 80% identity, at least90% identity, at least 95% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 1 and/or encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 2.

An exemplary CD28 hinge may comprise an amino acid sequence having atleast 80% identity, at least 90% identity, at least 95% identity, atleast 99% identity or is identical to SEQ ID NO: 3 and/or is encoded bya nucleotide sequence having at least 80% identity, at least 90%identity, at least 95% identity, at least 99% identity or is identicalto a sequence of SEQ ID NO: 4.

The transmembrane domain may comprise a TCR alpha transmembrane domain,a TCR beta transmembrane domain, or a CD3 zeta transmembrane domain,such as those disclosed by the present Inventors in PCT InternationalApplication No. PCT/US2020/045250, which is incorporated by reference.

A transmembrane domain can include one or more additional amino acidsadjacent to the transmembrane region, e.g., one or more amino acidsassociated with the extracellular region of the protein from which thetransmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to15 amino acids of the extracellular region) and/or one or moreadditional amino acids associated with the intracellular region of theprotein from which the transmembrane protein is derived (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellularregion).

The transmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins, e.g., tominimize interactions with other members of the receptor complex or tominimize interactions with other receptors. This can help, for example,to ensure that the receptors remain at a sufficient distance apart onthe surface of the immune cell to prevent blocking receptor inversion.

When present, the transmembrane domain may be a natural TCRtransmembrane domain, a natural transmembrane domain from a heterologousmembrane protein, or an artificial transmembrane domain. Thetransmembrane domain may be a membrane anchor domain. Withoutlimitation, a natural or artificial transmembrane domain may comprise ahydrophobic a helix of about 20 amino acids, often with positive chargesflanking the transmembrane segment.

The transmembrane domain may have one transmembrane segment or more thanone transmembrane segment. Prediction of transmembrane domains/segmentsmay be made using publicly available prediction tools, e.g., TMHMM(Krogh et al., Journal of Molecular Biology 2001, 305(3):567-580) andTMpred (Hoppe-Seyler, Hofmann & Stoffel Biol. Chem. 1993; 347: 166),which are incorporated by reference. Non-limiting examples of membraneanchor systems include platelet derived growth factor receptor (PDGFR)transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (addedpost-translationally to a signal sequence) and the like.

In certain aspects, transmembrane domain comprises a TCR alphatransmembrane domain. In some embodiments, the TCR alpha transmembranedomain comprises an amino acid sequence having at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 26 and/or is encoded by anucleotide sequence having at least 80% identity, at least 90% identity,at least 95% identity, at least 99% identity or is identical to asequence of SEQ ID NO: 27.

In some embodiments, the transmembrane domain comprises a TCR betatransmembrane domain. In some embodiments, the TCR beta transmembranedomain comprises an amino acid sequence having at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 28 or 35 and/or is encoded by anucleotide sequence having at least 80% identity, at least 90% identity,at least 95% identity, at least 99% identity or is identical to asequence of SEQ ID NO: 20 or 36.

In some embodiments, the transmembrane comprises a CD3 zetatransmembrane domain. In some embodiments, the CD3 zeta transmembranedomain comprises an amino acid sequence having at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 29.

In some embodiments, the CD3 zeta transmembrane domain comprises, orconsists essentially of, SEQ ID NO: 29. The disclosure provides fusionproteins comprising an intracellular domain. An “intracellular domain,”refers to an intracellular portion of a protein. The TCR intracellulardomain may comprise one or more domains capable of providing astimulatory signal to a transmembrane domain. The intracellular domainmay comprise a first intracellular domain capable of providing astimulatory signal and a second intracellular domain capable ofproviding a stimulatory signal. The intracellular domain may comprise afirst, second and third intracellular domain capable of providing astimulatory signal.

The intracellular domains capable of providing a stimulatory signal maybe selected from the group consisting of a CD28 molecule (CD28) domain,a LCK proto-oncogene, Src family tyrosine kinase (Lck) domain, a TNFreceptor superfamily member 9 (4-1BB) domain, a TNF receptor superfamilymember 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule(CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn)domain, a zeta chain of T cell receptor associated protein kinase 70(ZAP70) domain, a linker for activation of T cells (LAT) domain,lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCR beta,CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.

The TCR intracellular domain may comprise at least one intracellularsignaling domain. An intracellular signaling domain generates a signalthat promotes a function a cell, for example an immune effector functionof a TCR containing cell, e.g., a TCR-expressing T cell. In certainmethods and cells of the disclosure, the intracellular domain of thefusion proteins includes at least one intracellular signaling domain.For example, the intracellular domains of CD3 gamma, delta or epsiloncomprise signaling domains.

The extracellular domain, transmembrane domain and intracellular domainmay be isolated or derived from the same protein, for example T cellreceptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

Examples of intracellular domains for use in fusion proteins of thedisclosure include the cytoplasmic sequences of the TCR alpha, TCR beta,CD3 zeta, and 4-1BB, and the intracellular signaling co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement, as well as any derivative or variant of thesesequences and any recombinant sequence that has the same functionalcapability.

The intracellular signaling domain may comprise a primary intracellularsignaling domain. Exemplary primary intracellular signaling domainsinclude those derived from the proteins responsible for primarystimulation, or antigen dependent stimulation.

In some embodiments, the stimulatory domain comprises a CD28intracellular domain. In some embodiments, the CD28 intracellular domaincomprises an amino acid sequence having at least 85% identity, at least90% identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, at least 99% identity or is identicalto a sequence of SEQ ID NO: 37 and/or is encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 30.

In some embodiments, the stimulatory domain comprises a 4-IBBintracellular domain. In some embodiments, the 4-IBB intracellulardomain comprises an amino acid sequence having at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity or isidentical to a sequence of SEQ ID NO: 39 and/or is encoded by anucleotide sequence having at least 80% identity, at least 90% identity,at least 95% identity, at least 99% identity or is identical to asequence of SEQ ID NO: 40.

An intracellular signaling domain is generally responsible foractivation of at least one of the normal effector functions of theimmune cell in which the fusion protein has been introduced. The term“effector function” refers to a specialized function of a cell. Effectorfunction of a T cell, for example, may be cytolytic activity or helperactivity including the secretion of cytokines.

Thus, “intracellular signaling domain” refers to the portion of aprotein which transduces the effector function signal and directs thecell to perform a specialized function. While in some cases the entireintracellular signaling domain can be employed, in many cases it is notnecessary to use the entire intracellular signaling domain. To theextent that a truncated portion of the intracellular signaling domain isused, such truncated portion may be used in place of the intact chain aslong as it transduces the effector function signal. The termintracellular signaling domain is thus meant to include any truncatedportion of the intracellular signaling domain sufficient to transducethe effector function signal.

The intracellular domain may comprise the entirety or a portion of a CD3delta intracellular domain, a CD3 epsilon intracellular domain, a CD3gamma intracellular domain, or a CD3 zeta intracellular domain, such asthose disclosed by the present inventors in PCT InternationalApplication No. PCT/US2020/045250, which is incorporated by reference.

The intracellular domain may comprise a TCR alpha intracellular domainor a TCR beta intracellular domain, such as those disclosed by thepresent inventors in PCT International Application No.PCT/US2020/045250, incorporated by reference.

The intracellular signaling domain may comprise at least one stimulatoryintracellular domain. The intracellular signaling domain may comprise aprimary intracellular signaling domain, such as a CD3 delta, CD3 gammaand CD3 epsilon intracellular domain, and one additional stimulatoryintracellular domain, for example a co-stimulatory domain. Theintracellular signaling domain may comprise a primary intracellularsignaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilonintracellular domain, and two additional stimulatory intracellulardomains.

An exemplary CD3 delta intracellular domain may comprise, for example,an amino acid sequence having at least 85% identity, at least 90%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, at least 99% identity or is identicalto a sequence of SEQ ID NO: 30 and/or is encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 31.

An exemplary CD3 epsilon intracellular domain may comprise, for example,an amino acid sequence having at least 85% identity, at least 90%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, at least 99% identity or is identicalto a sequence of SEQ ID NO: 32 and/or is encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 19.

An exemplary CD3 gamma intracellular domain may comprise, for example,an amino acid sequence having at least 85% identity, at least 90%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, at least 99% identity or is identicalto a sequence of SEQ ID NO: 22 and/or is encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of SEQID NO: 9.

Exemplary co-stimulatory intracellular signaling domains include thosederived from proteins responsible for co-stimulatory signals, or antigenindependent stimulation.

The term “co-stimulatory molecule” refers to the cognate binding partneron a T-cell that specifically binds with a co-stimulatory ligand,thereby mediating a co-stimulatory response by the T-cell, such as, butnot limited to, proliferation. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors. Co-stimulatory molecules andtheir ligands are required for an efficient immune response.Co-stimulatory molecules include, but are not limited to an MHC class Imolecule, BTLA, a Toll ligand receptor, as well as DAP10, DAP12, CD30,LIGHT, OX40, CD2, CD27, CD S, ICAM-1, LFA-1 (CD11a/CD18) 4-1BB (CD137,TNF receptor superfamily member 9), and CD28 molecule (CD28).

A “co-stimulatory domain”, sometimes referred to as “a co-stimulatoryintracellular signaling domain” can be the intracellular portion of aco-stimulatory protein. A co-stimulatory domain can be a domain of aco-stimulatory protein that transduces the co-stimulatory signal. Aco-stimulatory protein can be represented in the following proteinfamilies: TNF receptor proteins, Immunoglobulin-like proteins, cytokinereceptors, integrins, signaling lymphocytic activation molecules (SLAMproteins), and activating NK cell receptors. Examples of such moleculesinclude CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR,HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically bindswith CD83, CD4, and the like. The co-stimulatory domain can comprise theentire intracellular portion, or the entire native intracellularsignaling domain, of the molecule from which it is derived, or afunctional fragment thereof.

The stimulatory domain may comprise a co-stimulatory domain. Theco-stimulatory domain may comprise a CD28 or 4-1BB co-stimulatorydomain. CD28 and 4-1BB are well characterized co-stimulatory moleculesrequired for full T cell activation and known to enhance T cell effectorfunction. For example, CD28 and 4-1BB have been utilized in chimericantigen receptors (CARs) to boost cytokine release, cytolytic function,and persistence over the first-generation CAR containing only the CD3zeta signaling domain. Likewise, inclusion of co-stimulatory domains,for example CD28 and 4-1BB domains, in engineered TCR can increase Tcell effector function and specifically allow co-stimulation in theabsence of co-stimulatory ligand, which is typically down-regulated onthe surface of tumor cells.

The stimulatory domain may comprise or be derived from a CD28intracellular domain or a 4-1BB intracellular domain, such as thosedisclosed by the present inventors in PCT International Application No.PCT/US2020/045250, which is incorporated herein by reference. Thedisclosure provides inhibitory intracellular domains which can be fusedto the transmembrane or intracellular domain of any of the TCR subunitsto generate a blocking TCR.

The inhibitory intracellular domain may comprise an immunoreceptortyrosine-based inhibitory motif (ITIM). The inhibitory intracellulardomain comprising an ITIM can be isolated or derived from an immunecheckpoint inhibitor such as CTLA-4 and PD-1.

Inhibitory domains can be isolated from human tumor necrosis factorrelated apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.

The inhibitory domain may comprise an intracellular domain, atransmembrane domain or a combination thereof. The inhibitory domain maycomprise an intracellular domain, a transmembrane domain, a hinge regionor a combination thereof. The inhibitory domain may comprise animmunoreceptor tyrosine-based inhibitory motif (ITIM). The inhibitorydomain comprising an ITIM can be isolated or derived from an immunecheckpoint inhibitor such as CTLA-4 and PD-1.

Inhibitory domains can be isolated from human tumor necrosis factorrelated apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.The inhibitory domain can be isolated or derived from a human protein,for example a human TRAIL receptor, CTLA-4, or PD-1 protein. In someembodiments, the TRAIL receptor comprises TR10A, TR10B or TR10D.

Endogenous TRAIL is expressed as a 281-amino acid type II trans-membraneprotein, which is anchored to the plasma membrane and presented on thecell surface. TRAIL is expressed by natural killer cells, which,following the establishment of cell-cell contacts, can induceTRAIL-dependent apoptosis in target cells. Physiologically, theTRAIL-signaling system was shown to be essential for immunesurveillance, for shaping the immune system through regulating T-helpercell 1 versus T-helper cell 2 as well as “helpless” CD8+ T-cell numbers,and for the suppression of spontaneous tumor formation.

The inhibitory domain may comprise an intracellular domain isolated orderived from a CD200 receptor. The cell surface glycoprotein CD200receptor 1 (Uniprot ref: Q8TD46) represents another example of aninhibitory intracellular domain of the present invention. Thisinhibitory receptor for the CD200/OX2 cell surface glycoprotein limitsinflammation by inhibiting the expression of proinflammatory moleculesincluding TNF-alpha, interferons, and inducible nitric oxide synthase(iNOS) in response to selected stimuli.

The inhibitory domain may be isolated or derived from killer cellimmunoglobulin like receptor, three Ig domains and long cytoplasmic tail2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domainsand long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin likereceptor B1 (LIR1), programmed cell death 1 (PD1), Fc gamma receptor IIB(FcgRIIB), killer cell lectin like receptor K1 (NKG2D), CTLA-4, a domaincontaining a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one orboth of the N and C terminal SH2 domains), or ZAP70 KI_K369A (kinaseinactive ZAP70).

The inhibitory domain can be isolated or derived from a human protein.

The blocking receptor may comprise a cytoplasmic domain andtransmembrane domain isolated or derived from the same protein, forexample an ITIM containing protein. The blocking receptor may comprise acytoplasmic domain, a transmembrane domain, and an extracellular domainor a portion thereof isolated or derived isolated or derived from thesame protein, for example an ITIM containing protein. The blockingreceptor may comprise a hinge region isolated or derived from isolatedor derived from the same protein as the intracellular domain and/ortransmembrane domain, for example an ITIM containing protein.

The blocking receptor may be a TCR comprising an inhibitory domain (aninhibitory TCR). The inhibitory TCR may comprise an inhibitoryintracellular domain and/or an inhibitory transmembrane domain. Theinhibitory intracellular domain can be fused to the intracellular domainof any one or more subunits of the TCR complex, including TCR alpha, TCRbeta, CD3 delta, CD3 gamma or CD3 epsilon, or a portion of any thereof.The inhibitory intracellular domain can be fused to the transmembranedomain of TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

The blocking receptor may comprise a hinge, transmembrane domain, and/oran intracellular domain derived from leukocyte immunoglobulin likereceptor B1 (LILRBI). The blocking receptor may comprise theintracellular domain of the protein phosphoprotein membrane anchor withglycosphingolipid microdomains 1 (PAG1) or a functional variant thereof,and optionally hinge, a transmembrane domain, and/or one or more furtherintracellular domains. The transmembrane domain may be the transmembranedomain of PAG1. The hinge, transmembrane domain, and/or a furtherintracellular domain may be from leukocyte immunoglobulin like receptorB1 (LILRB1), PAG1 or a combination thereof. Examples of such blockingreceptors have been disclosed by the Inventors of the present disclosurein U.S. Provisional Application Nos. 63/018,881 and 62/946,888 andPCT/US2021/030149, which are herein incorporated by reference in itsentirety.

In some embodiments of the receptors having one or more domains isolatedor derived from LILRB1, the one or more domains of LILRB1 comprise anamino acid sequence that is at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or is identical to asequence or subsequence of SEQ ID NO: 65, 77, 78, 79, 80, 81, 82, 83,84, or 85 and/or is encoded by a polynucleotide sequence that is atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or is identical to a sequence or subsequence ofSEQ ID NO: 66.

In various embodiments, an blocking receptor is provided, comprising apolypeptide, wherein the polypeptide comprises one or more of: an LILRB1hinge domain or functional fragment or variant thereof; an LILRB1transmembrane domain or a functional variant thereof; and an LILRB1intracellular domain or an intracellular domain comprising at least one,or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs),wherein each ITIM is independently selected from and/or includes asequence of SEQ ID NOS: 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

Assays

Provided herein are assays that can be used to measure the activity ofthe engineered receptors and immune cells disclosed herein.

Receptor activity may be assayed using a cell line engineered to expressa reporter of receptor activity such as a luciferase reporter. Exemplarycell lines include Jurkat T cells, although any suitable cell line knownin the art may be used. For example, Jurkat cells expressing aluciferase reporter under the control of an NFAT promoter can be used aseffector cells. Expression of luciferase by this cell line reflectsTCR-mediated signaling.

Nuclear factor of activated T cells (NFAT) is a family of transcriptionfactors shown to be important in immune response. The NFAT transcriptionfactor family consists of five members NFATc1, NFATc2, NFATc3, NFATc4,and NFAT5. NFAT plays a role in regulating inflammation. As used herein,an NFAT promoter is a promoter that is regulated (i.e., activated orrepressed) when NFAT is expressed in a cell. NFAT target promoters aredescribed in Badran, B. M. et al., (2002) J. Biological Chemistry, Vol.277: 47136-47148, incorporated herein by reference, and contain NFATconsensus sequences such as GGAAA.

The reporter cells can be transfected with each of the various fusionprotein constructs, combinations of fusion protein constructs orcontrols described herein. Expression of the fusion proteins in reportercells can be confirmed by using fluorescently labeled MHC tetramers, forexample Alexa Fluor 647-labeled NY-ESO-1-MHC tetramer, to detectexpression of the fusion protein.

To assay the activity of engineered receptors, target cells can beloaded with activating or blocking ligands prior to exposure to thecells comprising the reporter and the engineered receptor(s). Forexample, target cells can be loaded with ligands at least 12, 14, 16,18, 20, 22 or 24 hours prior to exposure to immune cells. Exemplarytarget cells include A375 cells, although any suitable cells known inthe art may be used. In some cases, target cells can be loaded withserially diluted concentrations of a ligand, such as NY-ESO-1 peptide.The immune cells can then be cocultured with target cells for a suitableperiod of time, for example 6 hours. Luciferase is then measured byluminescence reading after co-culture. Luciferase luminescence can benormalized to maximum and minimum intensity to allow comparison ofactivating peptide concentrations for each engineered receptorconstruct.

Provided herein are methods of determining the relative EC₅₀ ofengineered receptors of the disclosure. As used herein, “EC₅₀” refers tothe concentration of an inhibitor or agent to cause half the maximalresponse (or binding). Binding of the ligand, or probe to the engineeredreceptor can be measured by staining with labeled peptide or labeledpeptide-MHC complex, for example MHC:NY-ESO-1 pMHC complex conjugatedwith fluorophore. EC₅₀ can be obtained by nonlinear regression curvefitting of reporter signal with peptide titration. Probe binding andEC₅₀ can be normalized to the levels of benchmark TCR without a fusionprotein, e.g. NY-ESO-1 (clone 1G4).

Methods of assessing the effects of receptor activation on geneexpression are known in the art, and include the use of reporter genes,whose expression can be quantified. Reporter genes can be used foridentifying potentially transfected or transduced cells and forevaluating the functionality of regulatory sequences.

In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes apolypeptide whose expression is manifested by some easily detectableproperty, e.g., enzymatic activity. Expression of the reporter gene isassayed at a suitable time after the DNA has been introduced into therecipient cells. Suitable reporter genes may include genes encodingluciferase, beta-galactosidase, chloramphenicol acetyl transferase,secreted alkaline phosphatase, or the green fluorescent protein gene.See, e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82, which isincorporated herein by reference.

Suitable expression systems are well known and may be prepared usingknown techniques or obtained commercially. In general, the constructwith the minimal 5′ flanking region showing the highest level ofexpression of reporter gene is identified as the promoter. Such promoterregions may be linked to a reporter gene and used to evaluate agents forthe ability to modulate promoter-driven transcription. In exemplaryembodiments, an NFAT promoter operably linked to a reporter gene is usedto evaluate the expression of the receptors of the disclosure on NFATsignaling.

Exemplary assays have been disclosed by the present Inventors in PCTInternational Application Nos. PCT/US2019/037038, PCT/US2020/045250,PCT/US2020/045228, PCT/US2020/045373, and PCT/CA2016/051421, and U.S.Provisional Application Nos. 62/946,888, 62/934,419, 63/076,123,63/068,244, 63/068,249, 63/068,245, 63/068,246, 63/065,324, and63/037,975, which are each incorporated herein by reference.

Immune Cells

An immune cell can be a cell involved in the innate or adaptive(acquired) immune systems. Exemplary innate immune cells includephagocytic cells such as neutrophils, monocytes and macrophages, NaturalKiller (NK) cells, polymophonuclear leukocytes such as neutrophilseosinophils and basophils and mononuclear cells such as monocytes,macrophages and mast cells. Immune cells with roles in acquired immunityinclude lymphocytes such as T-cells and B-cells. An engineered immunecell of the present disclosure can be derived from an innate immune celland/or can be a modified innate immune cell.

A T cell is a type of lymphocyte that originates from a bone marrowprecursor that develops in the thymus gland. There are several distincttypes of T-cells which develop upon migration to the thymus, whichinclude, helper CD4+ T-cells, cytotoxic CD8+ T cells, memory T cells,regulatory CD4+ T-cells and stem memory T-cells. Different types of Tcells can be distinguished by the ordinarily skilled artisan based ontheir expression of markers. Methods of distinguishing between T celltypes will be readily apparent to the ordinarily skilled artisan.

The present disclosure also comprises methods of producing and modifyingthe engineered immune cells disclosed herein. The engineered immunecells of the present disclosure can be derived from any naturallyoccurring immune cell.

Methods of producing the disclosed immune cells may comprise introducingpolynucleotide encoding the activating and blocking receptors intocells, optionally using vectors. The resulting cells express thepolynucleotide encoding the receptors.

Methods transforming populations of immune cells, such as T cells, withvectors will be readily apparent to the person of ordinary skill in theart. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ Tcell negative isolation kit (Miltenyi), according to manufacturer'sinstructions. T cells can be cultured at a density of 1×10{circumflexover ( )}6 cells/mL in X-Vivo 15 media supplemented with 5% human ABserum and 1% Pen/strep in the presence of CD3/28 Dynabeads (1:1 cell tobead ratio) and 300 Units/mL of IL-2 (Miltenyi). After 2 days, T cellscan be transduced with viral vectors, such as lentiviral vectors usingmethods known in the art. In some embodiments, the viral vector istransduced at a multiplicity of infection (MOI) of 5. Cells can then becultured in IL-2 or other cytokines such as combinations of IL-7/15/21for an additional 5 days prior to enrichment.

Methods of isolating and culturing other populations of immune cells,such as B cells, or other populations of T cells, will be readilyapparent to the person of ordinary skill in the art. Although thismethod outlines a potential approach it should be noted that thesemethodologies are rapidly evolving. For example, high levels of viraltransduction of peripheral blood mononuclear cells can be achieved after5 days of growth to generate a >99% CD3+ highly transduced cellpopulation.

In some embodiments, the first and second receptors are encoded by asingle vector. Methods of encoding multiple polypeptides using a singlevector will be known to persons of ordinary skill in the art, andinclude, inter alia, encoding multiple polypeptides under control ofdifferent promoters, or, if a single promoter is used to controltranscription of multiple polypeptides, use of sequences encodinginternal ribosome entry sites (IRES) and/or self-cleaving peptides.Exemplary self-cleaving peptides include T2A, P2A, E2A and F2Aself-cleaving peptides. In some embodiments, the T2A self-cleavingpeptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 271). Insome embodiments, the P2A self-cleaving peptide comprises a sequence ofATNFSLLKQAGDVEENPGP (SEQ ID NO: 192). In some embodiments, the E2Aself-cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQID NO: 272). In some embodiments, the F2A selfcleaving peptide comprisesa sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 273).

Methods of activating and culturing populations immune cells comprisingthe receptors, polynucleotides, or vectors of the disclosure will bereadily apparent to the person of ordinary skill in the art.

Whether prior to or after genetic modification, the immune cells of thepresent disclosure can be activated and expanded generally using methodsas described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No.2006/0121005, each of which are incorporated herein by reference.

Immune cells of the instant disclosure can be expanded and activated invitro. Generally, the immune cells of the instant disclosure areexpanded in vitro by contact with a surface having an attached agentthat stimulates a CD3/TCR complex associated signal and a ligand thatstimulates a co-stimulatory molecule on the surface of the immune cells.Immune cell populations may be stimulated as described herein, such asby contact with an anti-CD3 antibody. For co-stimulation of an accessorymolecule on the surface of the immune cells, a ligand that binds theaccessory molecule can be used. For example, a population of T cells canbe contacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate to stimulate proliferation of the T cells. Inorder to stimulate proliferation of either CD4+ T cells or CD8+ T cells,an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples ofan anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,France) can be used as can other methods commonly known in the art, suchas in Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen etal., J. Exp. Med. 190(9):13191328, 1999; and Garland et al., J. ImmunolMeth. 227(1-2):53-63, 1999, each of which is incorporated herein byreference.

The primary stimulatory signal and the co-stimulatory signal for animmune cell of the disclosure may be provided by different protocols.For example, the agents providing each signal may be in solution orcoupled to a surface. When coupled to a surface, the agents may becoupled to the same surface (i.e., in “cis” formation) or to separatesurfaces (i.e., in “trans” formation). Alternatively, one agent may becoupled to a surface and the other agent in solution. The agentproviding the co-stimulatory signal may be bound to a cell surface andthe agent providing the primary activation signal is in solution orcoupled to a surface. Both agents can be in solution. The agents may bein soluble form, and then cross-linked to a surface, such as a cellexpressing Fc receptors or an antibody or other binding agent which willbind to the agents. U.S. Patent Application Publication Nos.2004/0101519 and 2006/0034810, which are incorporated herein byreference, disclose artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding immune cells of thepresent invention.

The two agents may be immobilized on beads, either on the same bead,i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, theagent providing the primary activation signal is an anti-CD3 antibody oran antigen-binding fragment thereof and the agent providing theco-stimulatory signal is an anti-CD28 antibody or antigen-bindingfragment thereof; and both agents are co-immobilized to the same bead inequivalent molecular amounts.

In certain methods of the disclosure, a 1:1 ratio of each antibody boundto the beads for CD4+ immune cell expansion and growth is used. Theratio of CD3:CD28 antibody bound to the beads may range from 100:1 to1:100, and all integer values there between. In one aspect of thepresent disclosure, more anti-CD28 antibody is bound to the particlesthan anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. Incertain methods of the disclosure, the ratio of anti CD28 antibody toanti CD3 antibody bound to the beads is greater than 2:1.

Ratios of particles to cells from 1:500 to 500:1, and any integer valuesin between, may be used to stimulate immune cells, such as T cells, orother target cells. As those of ordinary skill in the art can readilyappreciate, the ratio of particles to cells may depend on particle sizerelative to the target cell. For example, small sized beads may onlybind a few cells, while larger beads can bind many.

In certain methods of the disclosure, the ratio of cells to particlesranges from 1:100 to 100:1, and any integer values in-between, can beused to stimulate the immune cells. In certain methods of thedisclosure, the ratio comprises 1:9 to 9:1 and any integer values inbetween. In certain methods, a ratio of 1:1 cells to beads may be used.One of skill in the art will appreciate that a variety of other ratiosmay be suitable for use in the present invention. In particular, theratios used will vary depending on particle size and on cell size andtype.

In further methods of the present disclosure, the immune cells, such asT cells, are combined with agent-coated beads, the beads and the cellsare subsequently separated, and then the cells are cultured.Alternatively, prior to culture, the agent-coated beads and cells arenot separated, but are cultured together. The beads and cells mayinitially be concentrated by application of a force, such as a magneticforce, resulting in increased ligation of cell surface markers, therebyinducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached tocontact the immune cells. The cells (for example, CD4+ T cells) andbeads (for example, DYNABEADS CD3/CD28 T paramagnetic beads at a ratioof 1:1) are combined in a buffer. Again, those of ordinary skill in theart can readily appreciate any cell concentration may be used. Incertain methods, it may be desirable to significantly decrease thevolume in which particles and cells are mixed together (i.e., increasethe concentration of cells), to ensure maximum contact of cells andparticles. For example, a concentration of about 2 billion cells/ml canbe used. Alternatively, greater than 100 million cells/ml can be used. Aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml can be used. A concentration of cells from 75, 80, 85, 90, 95,or 100 million cells/ml can be used. Concentrations of 125 or 150million cells/ml can be used. In certain methods, cells are cultured ata density of 1×10⁶ cells/mL.

The mixture may be cultured for several hours (about 3 hours) to about14 days or any hourly integer value in between. The beads and immunecells may be cultured together for 2-3 days. Conditions appropriate forimmune cell culture include an appropriate media (e.g., MinimalEssential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that maycontain factors necessary for proliferation and viability, includingserum (e.g., fetal bovine or human serum), interleukin-2 (IL-2),insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-αor any other additives for the growth of cells known to the skilledartisan. Other additives for the growth of cells include, but are notlimited to, surfactant, plasmanate, and reducing agents such asN-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640,AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, withadded amino acids, sodium pyruvate, and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of immune cells. The media may compriseXVIVO-15 media supplemented with 5% human A/B serum, 1%penicillin/streptomycin (pen/strep) and 300 Units/ml of IL-2 (Miltenyi).

The engineered immune cells can be maintained under conditions necessaryto support growth, for example, an appropriate temperature (e.g., 37°C.) and atmosphere (e.g., air plus 5% CO2).

Immune cells comprising receptors of the present disclosure may beautologous. Prior to expansion and genetic modification, a source ofimmune cells can obtained from a subject, such as a human patient.Immune cells, such as T cells, can be obtained from a number of sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors.

In certain methods of the disclosure, any number of immune cell linesavailable in the art, may be used. Immune cells can be obtained from aunit of blood collected from a subject using any number of techniquesknown to the skilled artisan, such as Ficoll™ separation.

Cells from the circulating blood of an individual can be obtained byapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. Cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.The cells can be washed with phosphate buffered saline (PBS). The washsolution may lack calcium and magnesium or may lack many, if not all,divalent cations. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

Immune cells, such as T cells, can be isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. Specific subpopulations of immunecells, such as T cells, B cells, or CD4+ T cells can be further isolatedby positive or negative selection techniques. For example, T cells canbe isolated by incubation with anti-CD4-conjugated beads, for a timeperiod sufficient for positive selection of the desired T cells.

Enrichment of an immune cell population, such as a T cell population, bynegative selection can be accomplished with a combination of antibodiesdirected to surface markers unique to the negatively selected cells. Onemethod is cell sorting and/or selection via negative magneticimmune-adherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail typically includes antibodiesto CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

For isolation of a desired population of immune cells by positive ornegative selection, the concentration of cells and surface (e.g.,particles such as beads) can be varied. In certain embodiments, it maybe desirable to significantly decrease the volume in which beads andcells are mixed together (i.e., increase the concentration of cells), toensure maximum contact of cells and beads.

The cells may be incubated on a rotator for varying lengths of time atvarying speeds at either 2-10° C. or at room temperature.

T cells for stimulation, or PBMCs from which immune cells such as Tcells are isolated, can also be frozen after a washing step. Wishing notto be bound by theory, the freeze and subsequent thaw step provides amore uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

Exemplary immune cells and methods for producing the same include thosethat have been disclosed by the present Inventors in PCT InternationalApplication Nos. PCT/US2019/037038, PCT/US2020/045250,PCT/US2020/045228, PCT/US2020/045373, and PCT/CA2016/051421, and U.S.Provisional Application Nos. 62/946,888, 62/934,419, 63/076,123,63/068,244, 63/068,249, 63/068,245, 63/068,246, 63/065,324, and63/037,975, which are each incorporated herein by reference.

Target Ligands

The disclosure provides receptors comprising extracellular ligandbinding domains. The ligand may be an antigen and the ligand bindingdomain may be an antigen binding domain.

Any suitable ligand binding domain is envisaged as within the scope ofthe receptors described herein.

The ligand binding domain of the activating or blocking receptors maycomprise an antigen binding domain comprises an antibody fragment, a VPonly domain, a linear antibody, a single-chain variable fragment (scFv),or a single domain antibody (sdAb).

The receptors may each comprise two polypeptides each having a part of aligand-binding domain (e.g. cognates of a heterodimeric LDB, such as aTCRα/β- or Fab-based LBD). The disclosure further provides receptorshaving two polypeptides, each having a part of a ligand-binding domain(e.g. cognates of a heterodimeric LDB, such as a TCRα/β- or Fab-basedLBD) and one part of the ligand binding domain is fused to a hinge ortransmembrane domain, while the other part of the ligand binding domainhas no intracellular domain. Further variations include receptors whereeach polypeptide has a hinge domain, and where each polypeptide has ahinge and transmembrane domain. Some receptors may not have a hingedomain.

The ligand binding domain of the receptors may comprise a Fab fragmentof an antibody.

Receptors of the present disclosure may comprise a first polypeptidethat comprises an antigen-binding fragment of the heavy chain of anantibody and an intracellular domain, and a second polypeptide of thereceptor comprises an antigen-binding fragment of the light chain of theantibody. Alternatively, the first polypeptide may comprise anantigen-binding fragment of the light chain of the antibody and theintracellular domain, and the second polypeptide comprises anantigen-binding fragment of the heavy chain of the antibody.

The blocking and/or activating receptors may comprise an extracellularfragment of a T cell receptor (TCR).

Any macromolecule, including virtually all proteins or peptides, canserve as an antigen for the receptors described herein. Antigens can bederived from recombinant or genomic DNA. Any DNA, which comprises anucleotide sequence or a partial nucleotide sequence encoding a proteinthat elicits an immune response, encodes an antigen. An antigen need notbe encoded solely by a full-length nucleotide sequence of a gene. Anantigen need not be encoded by a gene. An antigen can be generatedsynthesized or can be derived from a biological sample, or might bemacromolecule besides a polypeptide. Such a biological sample caninclude, but is not limited to a tissue sample, a tumor sample, a cellor a fluid with other biological components.

In the engineered receptors of the present disclosure, theantigen-binding domain may specifically bind to a target selected frometiolate receptor, αvββ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20,CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138,CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, ErbB2 (HER2), ErbB3(Her3), ErbB4 (Her4), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetalacetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11R,IL13R-a2, KDR, κ light chain, λ, light chain, LeY, LI CAM, MAGE-A1,mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands,Notch1, Notch2/3, NYESO-1, PRAIVIE, PSCA, PSMA, Survivin, TAG-72, TEMs,TERT, VEGFR2, and ROR1.

The antigen-binding domain may specifically bind peptide MHC (pMHC) asthe antigen. Exemplary pMHC antigens include, but are not limited to,MAGE-A3 pMHC (e.g., FLWGPRALV and MPKVAELVHFL peptides), HPV E6 pMHC(e.g., TIHDIILECV peptide), HPV E7 pMHC (e.g., YMLDLQPET peptide) andNY-ESO-1 pMHC (e.g., LLEFYLAMPFA or SLLMWITQV peptides).

The antigen-binding domain may specifically bind to a target selectedfrom CD33, CD38, a human leukocyte antigen (HLA), an organ specificantigen, a blood-brain barrier specific antigen, anEpithelial-mesenchymal transition (EMT) antigen, E-cadherin,cytokeratin, Opioid-binding protein/cell adhesion molecule (OPCML),HYLA2, Deleted in Colorectal Carcinoma (DCC), Scaffold/Matrix attachmentregion-binding protein 1 (SMAR1), cell surface carbohydrate and mucintype 0-glycan.

The antigen-binding domain of the blocking receptor may specificallybind to an antigen from a gene with high, homogeneous surface expressionacross tissues. High, homogeneous surface expression across tissuesallows the blocking receptor to deliver a large, even inhibitory signal.The antigen may be encoded by a gene that is absent or polymorphic in inmany tumors.

Methods of distinguishing the differential expression of blockingligands (e.g., antigens) between target and non-target cells can be usedin methods and systems of the invention. For example, the presence orabsence of blocking ligands in nontarget and target cells can be assayedby immunohistochemistry with an antibody that binds to the inhibitorligand, followed by microscopy or FACS, RNA expression profiling oftarget cells and non-target cells, or DNA sequencing of non-target andtarget cells to determine if the genomic locus of the blocking ligandcomprises mutations in either the target or non-target cells.

Homozygous deletions in primary tumors are rare and small, and thereforeunlikely to yield blocking ligand candidates. For example, in ananalysis of 2218 primary tumors across 21 human cancer types, the top 4candidates were CDKN2A, RB1, PTEN and N3PB2. However, CDKN2A (P16) wasdeleted in only 5% homozygous deletion across all cancers. HomozygousHLA-A deletions were found in less than 0.2% of cancers in Cheng et al.,Nature Comm. 8:1221 (2017), incorporated herein by reference. Incontrast, deletion of a single copy of gene in cancer cells due to lossof hemizygosity occurs far more frequently.

Thus, the blocking ligand may comprise an allele of a gene that is lostin target cells due to loss of heterozygosity, and the target cells maycomprise cancer cells. Cancer cells undergo frequent genomerearrangements, including duplication and deletions. These deletions canlead to the deletion of one copy of one or more genes in the cancercells.

Loss of heterozygosity (LOH) refers to a genetic change, whereby one ofthe two alleles in the genome of a cell or cells is deleted, leaving asingle mono-allelic (hemizygous) locus.

The blocking ligand may comprise an HLA class I allele. The majorhistocompatibility complex (MHC) class I is a gene complex that encodesproteins that display antigens to cells of the immune system, triggeringimmune response. The Human Leukocyte Antigens (HLAs) corresponding toMHC class I are HLA-A, HLA-B and HLA-C.

The blocking ligand may comprise an HLA class I allele. The blockingligand may comprise an allele of HLA class I that is lost in a targetcell through LOH. HLA-A is a group of human leukocyte antigens (HLA) ofthe major histocompatibility complex (MHC) that are encoded by the HLA-Alocus. HLA-A is one of three major types of human MHC class I cellsurface receptors. The receptor is a heterodimer comprising a heavy αchain and smaller β chain. The α chain is encoded by a variant of HLA-A,while the β chain (β2-microglobulin) is an invariant. There are severalthousand variant HLA-A alleles, all of which fall within the scope ofthe instant disclosure.

The blocking ligand may comprise an HLA-B allele. The HLA-B gene hasmany possible variations (alleles). Hundreds of versions (alleles) ofthe HLA-B gene are known, each of which is given a particular number(such as HLAB27).

The blocking ligand may comprise an HLA-C allele. HLA-C belongs to theHLA class I heavy chain paralogues. This class I molecule is aheterodimer consisting of a heavy chain and a light chain (beta-2microglobulin). Over one hundred HLA-C alleles have been described.

The HLA class I allele may have broad or ubiquitous RNA expression. TheHLA class I allele may have a known, or generally high minor allelefrequency. The HLA class I allele may not require a peptide-MHC antigen,for example when the HLA class I allele is recognized by a pan-HLAligand binding domain.

The blocking ligand may comprise an HLA-A allele. The HLA-A allele maycomprise HLA-A*02. Various single variable domains known in the art ordisclosed herein that bind to and recognize HLA-A*02 are suitable foruse in the present disclosure. Such scFvs include, for example andwithout limitation the following mouse and humanized scFv antibodiesthat bind HLA-A*02 in a peptide independent manner.

The blocking ligand (e.g., an antigen) may comprise a minorhistocompatibility antigen (MiHA). The inhibitor ligand may comprise anallele of a MiHA that is lost in a target cell through LOH.

MiHAs are peptides derived from proteins that contain nonsynonymousdifferences between alleles and are displayed by common HLA alleles. Thenonsynonymous differences can arise from SNPs, deletions, frameshiftmutations or insertions in the coding sequence of the gene encoding theMiHA. Exemplary MiHAs can be about 9-12 amino acids in length and canbind to WIC class I and/or WIC class II proteins. Binding of the TCR tothe MHC complex displaying the MiHA can activate T cells. The geneticand immunological properties of MiHAs will be known to the person ofordinary skill in the art. Candidate MiHAs are known peptides presentedby known HLA class I alleles, are known to elicit T cell responses inthe clinic (for example, in graft versus host disease, or transplantrejection), and allow for patient selection by simple SNP genotyping.

The MiHA may have broad or ubiquitous RNA expression. The MiHA may havehigh minor allele frequency. The MiHA may comprise a peptide derivedfrom a Y chromosome gene.

The blocking ligand may comprise a Y chromosome gene, i.e. peptideencoded by a gene on the Y chromosome. The blocking ligand may comprisea peptide encoded by a Y chromosome gene that is lost in target cellsthrough loss of Y chromosome (LoY). For example, about a third of thecharacterized MiHAs come from the Y chromosome. The Y chromosomecontains over 200 protein coding genes, all of which are envisaged aswithin the scope of the instant disclosure.

As used herein, “loss of Y”, or “LoY” refers a genetic change thatoccurs at high frequency in tumors whereby part or all of the Ychromosome is deleted, leading to a loss of Y chromosome encodedgene(s).

Loss of Y chromosome is known to occur in certain cancers. For example,there is a reported 40% somatic loss of Y chromosome in renal clear cellcancers (Arseneault et al., Sci. Rep. 7: 44876 (2017)). Similarly,clonal loss of the Y chromosome was reported in 5 out of 31 in malebreast cancer subjects in Wong et al., Oncotarget 6(42):44927-40 (2015),incorporated herein by reference. Loss of the Y chromosome in tumorsfrom male patients has been described as a “consistent feature” of headand neck cancer patients, as in el-Naggar et al., Am J Clin Pathol105(1):102-8 (1996), incorporated herein by reference. Further, Ychromosome loss was associated with X chromosome disomy in four of sevenmale patients with gastric cancer in Saal et al., Virchows Arch B CellPathol (1993), incorporated herein by reference. Thus, Y chromosomegenes can be lost in a variety of cancers, and can be used as blockingligands with the engineered receptors of the instant disclosuretargeting cancer cells.

The activating ligand may be a transferrin receptor (TFRC). Humantransferrin receptor is described in NCBI record No. AAA61153.1, thecontents of which are incorporated herein by reference.

The activating ligand may be a tumor specific antigen (TSA). The tumorspecific antigen may be mesothelin (MSLN), CEACAMS or EGFR. The TSA maybe MSLN, CEA, EGFR, DLL4, CA125, GD2, ROR1 or HER2/NEU. The activatingligand may be a pan-HLA ligand, and the activating receptor ligandbinding domain is a pan-HLA binding domain, i.e. a binding domain thatbinds to and recognizes an antigenic determinant shared among HLA Iproducts, such as the HLA A, B and C loci. The activating ligand mayalso be another class I gene product; e.g., antigens encoded by HLA-E orF. Various single variable domains known in the art are suitable for usein embodiments. Such scFvs include, for example and without limitationthe following mouse and humanized pan-HLA scFv antibodies. An exemplarypan-HLA ligand is W6/32, which recognizes a conformational epitope,reacting with HLA class I alpha3 and alpha2 domains. Further exemplaryantibodies with broad HLA binding are known in the art and include HC-10and TFL-006. Exemplary activating ligands and activating receptor ligandbinding domains have been disclosed by the Inventors of the presentdisclosure in U.S. Provisional Application No. 63/018,881, which isherein incorporated by reference in its entirety.

Exemplary ligands and ligand binding domains of activating and blockingreceptors include those that have been disclosed by the presentInventors in PCT International Application Nos. PCT/US2019/037038,PCT/US2020/045250, PCT/US2020/045228, PCT/US2020/045373, andPCT/CA2016/051421, and U.S. Provisional Application Nos. 62/946,888,62/934,419, 63/076,123, 63/068,244, 63/068,249, 63/068,245, 63/068,246,63/065,324, and 63/037,975, which are each incorporated herein byreference.

For example, in some embodiments of the immune cells of the disclosure,a first/activating ligand is EGFR or a peptide antigen thereof, and thefirst/activating ligand binding domain comprises a sequence of SEQ IDNO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, or SEQID NO: 391, or a sequence having at least 90%, at least 95% or at least99% identity thereto. In some embodiments, the first ligand bindingdomain comprises CDRs selected from SEQ ID NOs: 131-166.

In some embodiments, the activator ligand is EGFR or a peptide antigenthereof, and the activator ligand binding domain comprises an EGFRbinding domain. In some embodiments, the EGFR ligand binding domaincomprises an ScFv domain. In some embodiments, the EGFR ligand bindingdomain comprises a sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ IDNO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114,SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391. In some embodiments,the EGFR ligand binding domain comprises a sequence at least 90%, atleast 95% or at least 99% identical to SEQ ID NO: 102, SEQ ID NO: 104,SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ IDNO: 114, SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391. In someembodiments, the EGFR ligand binding domain is encoded by a sequencecomprising SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117 orSEQ ID NO: 119. In some embodiments, the EGFR ligand binding domain isencoded by a sequence having at least 80% identity, at least 85%identity, at least 90% identity, at least 95% identity or at least 99%identity to a sequence of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQID NO: 117 or SEQ ID NO: 119.

In some embodiments, the activator ligand is EGFR or a peptide antigenthereof, and the activator ligand binding domain comprises an EGFRligand binding domain. In some embodiments, the EGFR binding domaincomprises a VH and/or a VL domain selected from the group disclosed inTable 2 or a sequence having at least 90% identity thereto. In someembodiments, the EGFR ligand binding domain comprises a VH domainselected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122,SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128 and SEQ ID NO: 130. Insome embodiments, the EGFR ligand binding domain comprises a VH selectedfrom the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:124, SEQ ID NO: 126, SEQ ID NO: 128 and SEQ ID NO: 130 or a sequencehaving at least 90%, at least 95% or at least 99% identity thereto. Insome embodiments, the EGFR ligand binding domain comprises a VL domainselected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123,SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129 and SEQ ID NO: 131. Insome embodiments, the EGFR ligand binding domain comprises a VH selectedfrom the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:125, SEQ ID NO: 127, SEQ ID NO: 129 and SEQ ID NO: 131 or a sequencehaving at least 90%, at least 95% or at least 99% identity thereto.

In some embodiments, the activator ligand is EGFR or a peptide antigenthereof, and the activator ligand binding domain is an EGFR ligandbinding domain. In some embodiments, the EGFR binding domain comprisescomplementarity determining region (CDRs) selected from the group ofCDRs disclosed in Table 3. In some embodiments, the EGFR ligand bindingdomain comprises CDRs having at least 95% sequence identity to CDRsdisclosed in Table 3. In some embodiments, the EGFR ligand bindingdomain comprises CDRs selected from SEQ ID NOs: 131-166. In someembodiments, the EGFR ligand binding domain comprises a heavy chain CDR1 (CDR HI) selected from the group consisting of SEQ ID NOs: 132-137. Insome embodiments, the EGFR ligand binding domain comprises a heavy chainCDR 2 (CDR H2) selected from the group consisting of SEQ ID NOs:138-143. In some embodiments, the EGFR ligand binding domain comprises aheavy chain CDR 3 (CDR H3) selected from the group consisting of SEQ IDNOs: 144-149. In some embodiments, the EGFR ligand binding domaincomprises a light chain CDR 1 (CDR LI) selected from the groupconsisting of SEQ ID NOs: 150-155. In some embodiments, the EGFR ligandbinding domain comprises a light chain CDR 2 (CDR L2) selected from thegroup consisting of SEQ ID NOs: 156-160. In some embodiments, the EGFRligand binding domain comprises a light chain CDR 3 (CDR L3) selectedfrom the group consisting of SEQ ID NOs: 161-166. In some embodiments,the EGFR ligand binding domain comprises a CDR HI selected from SEQ IDNOs: 132-137, a CDR H2 selected from SEQ ID NOs: 138-143, a CDR H3selected from SEQ ID NOs: 144-149, a CDR LI selected from SEQ ID NOs:150-155, a CDR L2 selected from SEQ ID NOs: 156-160, and a CDR L3selected from SEQ ID NOs: 156-160. [0177] Table 3. EGFR antigen bindingdomain CDRs.

In some embodiments of the immune cells of the disclosure, thefirst/activating ligand is MSLN or a peptide antigen thereof. In someembodiments, the first/activating ligand binding domain comprises asequence of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90 or SEQ P) NO:92, or a sequence having at least 90%, at least 95% or at least 99%identity thereto. In some embodiments, the MSLN ligand binding domain isencoded by a sequence comprising SEQ ID NO: 87, SEQ ID NO: 89, SEQ IDNO: 91 or SEQ ID NO: 93. In some embodiments, the MSLN ligand bindingdomain is encoded by a sequence having at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity or at least99% identity to a sequence of SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO:91 or SEQ ID NO: 93.

In some embodiments of the immune cells of the disclosure, thefirst/activating ligand is CEA or a peptide antigen thereof. In someembodiments, the first/activating ligand binding domain comprises SEQ IDNO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282,SEQ ID NO: 284, or SEQ ID NO: 286, or a sequence having at least 90%, atleast 95% or at least 99% identity thereto. In some embodiments, thefirst/activating ligand binding domain comprises CDRs selected from SEQID NOs: 294-302. In some embodiments, the CEA ligand binding domain isencoded by a sequence having at least 80% identity, at least 85%identity, at least 90% identity, at least 95% identity or at least 99%identity to a sequence of SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99,SEQ ID NO: 101, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287.

In some embodiments, the activator ligand is CEA or a peptide antigenthereof, and the activator ligand binding domain comprises a CEA bindingdomain. In some embodiments, the CEA ligand binding domain comprises aCDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ IDNO: 295), a CDR-H3 of WDF AYYVEAMD Y (SEQ ID NO: 296) or WDFAHYFQTMDY(SEQ ID NO: 297), a CDR-L1 of KASQNVGTNV A (SEQ ID NO: 298) orKASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRYS (SEQ ID NO: 300) orSASYRKR (SEQ ID NO: 301), and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302)or sequences having at least 85% or at least 95% identity thereto. Insome embodiments, a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO:294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMD Y (SEQ ID NO: 296) or WDFAHYFQTMDY (SEQ ID NO: 297), a CDR-L1of KASQNVGTNV A (SEQ ID NO: 298) or KASAAVGTYVA (SEQ ID NO: 299), aCDR-L2 of SASYRYS (SEQ ID NO: 300) or SASYRKR (SEQ ID NO: 301) and aCDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302).

In some embodiments, a CEA binding domain comprises a CDR-H1 of EFGMN(SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), aCDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296), a CDR-L1 of KASQNVGTNV A (SEQID NO: 298), a CDR-L2 of SASYRYS (SEQ ID NO: 300) and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA ScFv comprises aCDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ IDNO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296), a CDR-L1 ofKASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRKR, and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA binding domaincomprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAHYFQTMD Y (SEQ ID NO: 297), aCDR-L1 of KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRKR, and aCDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302).

In some embodiments, the activator ligand is CEA or a peptide antigenthereof, and the activator receptor is a CEA CAR In some embodiments,the CEA CAR comprises sequence at least 90%, at least 95% or at least99% identical to SEQ ID NO: 288, SEQ ID NO: 290 or SEQ ID NO: 292. Insome embodiments, the CEA CAR comprises or consists essentially of SEQID NO: 288, SEQ ID NO: 290 or SEQ ID NO: 292. In some embodiments, theCEA CAR is encoded by a sequence comprising or consisting essentially ofSEQ ID NO: 289, SEQ ID NO: 291 or SEQ ID NO: 293. In some embodiments,the CEA CAR is encoded by a sequence having at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity or atleast 99% identity to SEQ ID NO: 289, SEQ ID NO: 291 or SEQ ID NO: 293.

In some embodiments of the immune cells of the disclosure, thefirst/activating ligand is CD19 or a peptide antigen thereof, and thefirst ligand binding domain comprises SEQ ID NO: 275 or SEQ ID NO: 277,or a sequence having at least 90%, at least 95% or at least 99% identitythereto.

In some embodiments of the immune cells of the disclosure, thefirst/activating ligand is a pan-HLA ligand. In some embodiments, thefirst ligand binding domain comprises a sequence of SEQ ID NO: 167, SEQID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ IDNO: 177, or a sequence having at least 90%, at least 95% or at least 99%identity thereto.

In some embodiments of the immune cells of the disclosure, thesecond/blocking ligand comprises HA-1. In some embodiments, and whereinthe second/blocking ligand binding domain comprises a TCR alpha variabledomain comprising SEQ ID NO: 199 or a sequence having at least 90%, atleast 95%, or at least 99% identity thereto, and a TCR beta variabledomain comprising SEQ ID NO: 200 or a sequence having at least 90%, atleast 95%, or at least 99% identity thereto. In some embodiments, thesecond/blocking ligand binding domain comprises a TCR alpha variabledomain comprising SEQ ID NO: 199, and a TCR beta variable domaincomprising SEQ ID NO: 200.

In some embodiments of the immune cells of the disclosure, thesecond/blocking ligand comprises an HLA-A*02 allele. In someembodiments, the second/blocking ligand binding domain comprises any oneof SEQ ID NOs: 53-64 or a sequence having at least 90%, at least 95%, orat least 99% identity thereto. In some embodiments, the second/blockingligand binding domain comprises CDRs selected from SEQ ID NOs: 41-52.

In some embodiments of the inhibitory/blocking receptors of thedisclosure, the extracellular ligand binding domain has a higheraffinity for an HA-1 (H) peptide of VLHDDLLEA (SEQ ID NO: 191) than foran HA-1(R) peptide of VLRDDLLEA (SEQ ID NO: 266). In some embodiments,the inhibitory/blocking receptor is activated by the HA-1(H) peptide ofVLHDDLLEA (SEQ ID NO: 191) and is not activated, or activated to alesser extent, by the HA-1(R) peptide of VLRDDLLEA (SEQ ID NO: 266). Insome embodiments, the extracellular ligand binding domain comprises aTCR alpha variable domain comprising SEQ ID NO: 199 or a sequence havingat least 90%, at least 95%, or at least 99% identity thereto, and a TCRbeta variable domain comprising SEQ ID NO: 200 or a sequence having atleast 90%, at least 95%, or at least 99% identity thereto. In someembodiments, the extracellular ligand binding domain comprises a TCRalpha variable domain comprising SEQ ID NO: 199 and a TCR beta variabledomain comprising SEQ ID NO: 200.

In some embodiments, the activator ligand is pan-HLA ligand, and theactivator ligand binding domain comprises a pan-HLA ligand bindingdomain. In some embodiments, the pan-HLA ligand binding domain comprisesan ScFv domain. In some embodiments, the pan-HLA ligand binding domaincomprises a sequence of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171,SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments,the pan-HLA ligand binding domain comprises a sequence at least 90%, atleast 95% or at least 99% identical to SEQ ID NO: 167, SEQ ID NO: 169,SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. Insome embodiments, the pan-HLA ligand binding domain is encoded by asequence comprising SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178. In some embodiments, thepan-HLA ligand binding domain is encoded by a sequence having at least80% identity, at least 85% identity, at least 90% identity, at least 95%identity or at least 99% identity to a sequence of SEQ ID NO: 168, SEQID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ IDNO: 178. [0181] In some embodiments, the activator ligand is CD19molecule (CD19) or a peptide antigen thereof, and the activator ligandbinding domain comprises a CD 19 ligand binding domain. In someembodiments, the CD 19 ligand binding domain comprises an ScFv domain.In some embodiments, the CD 19 ligand binding domain comprises asequence at least 90%, at least 95% or at least 99% identical to SEQ IDNO: 275 or SEQ ID NO: 277. In some embodiments, the CD-19 ligand bindingdomain comprises a sequence of SEQ ID NO: 275 or SEQ ID NO: 277. In someembodiments, the CD19 ligand binding domain is encoded by a sequencecomprising SEQ ID NO: 276, or SEQ ID NO: 278. In some embodiments, theCD19 ligand binding domain is encoded by a sequence having at least 80%identity, at least 85% identity, at least 90% identity, at least 95%identity or at least 99% identity to a sequence of SEQ ID NO: 276 or SEQID NO: 278.

In some embodiments, activator ligand is CD19 molecule (CD19) or apeptide antigen thereof, and the activator receptor is a CAR In someembodiments, the CD 19 CAR comprises a sequence at least 90%, at least95% or at least 99% identical to SEQ ID NO: 279 or SEQ ID NO: 281. Insome embodiments, the CD 19 CAR comprises or consists essentially of SEQID NO: 279 or SEQ ID NO: 281. In some embodiments, the CD19 CAR isencoded by a sequence having at least 80% identity, at least 85%identity, at least 90% identity, at least 95% identity or at least 99%identity to a sequence of SEQ ID NO: 280 or SEQ P) NO: 390. In someembodiments, the CD19 CAR is encoded by a sequence comprising orconsisting essentially of SEQ ID NO: 280 or SEQ ID NO: 390.

In some embodiments, the one or more ligand comprises an HLA-A allele.In some embodiments the HLA-A allele comprises HLA-A*02. Various singlevariable domains known in the art or disclosed herein that bind to andrecognize HLA-A*02 are suitable for use in embodiments. Such scFvsinclude, for example and without limitation, the following mouse andhumanized scFv antibodies that bind HLA-A*02 in a peptide-independentway, which include binding domains having at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity or atleast 99% identity to any one of SEQ ID NOS: 179-190.

In some embodiments, the scFv comprises the complementarity determinedregions (CDRs) of any one of SEQ ID NOS: 41-52. In some embodiments, thescFv comprises a sequence at least 95% identical to any one of SEQ P)NOS: 41-52. In some embodiments, the scFv comprises a sequence identicalto any one of SEQ ID NOS: 41-52. In some embodiments, the heavy chain ofthe antibody comprises the heavy chain CDRs of any one of SEQ ID NOS:53-64, and wherein the light chain of the antibody comprises the lightchain CDRs of any one of SEQ ID NOS: 53-64. In some embodiments, theheavy chain of the antibody comprises a sequence at least 95% identicalto the heavy chain portion of any one of SEQ ID NOS: 53-64, and whereinthe light chain of the antibody comprises a sequence at least 95%identical to the light chain portion of any one of SEQ ID NOS: 53-64.[0209] In some embodiments, the heavy chain of the antibody comprises asequence identical to the heavy chain portion of any one of SEQ ID NOS:53-64, and wherein the light chain of the antibody comprises a sequenceidentical to the light chain portion of any one of SEQ ID NOS: 53-64.

In some embodiments, a ligand as used herein comprises a minorhistocompatibility antigen (MiHA). In some embodiments, the blockingligand comprises an allele of a MiHA that is lost in a target cellthrough LOH. Exemplary, but non-limiting, examples of MiHAs that areenvisaged as within the scope of the instant invention include thosehaving the sequence of any one of SEQ ID NOS: 273, 303-325, 327-356,358-389, 34, and 23-25.

Exemplary ligand binding domains that selectively bind to HA-1 variant Hpeptide (VLHDDLLEA (SEQ ID NO: 191)) include the sequences of SEQ ID NO:194, 201, 202, 196, and 198. TCR alpha and TCR beta sequences in SEQ IDNO: 193 are separated by a P2A self-cleaving polypeptide of sequenceATNFSLLKQAGDVEENPGP (SEQ ID NO: 192) with an N terminal GSG linker.

In some embodiments, the TCR alpha and TCR beta variable domainsseparated by a self-cleaving polypeptide sequence comprise SEQ ID NO:193, or a sequence having at least 90%, at least 95%, or at least 99%identity thereto. In some embodiments, the TCR alpha and TCR betavariable domains are encoded by a sequence of SEQ ID NO: 194, or asequence having at least 80% identity, at least 90%, at least 95%, or atleast 99% identity thereto. In some embodiments, the TCR alpha variabledomain comprises SEQ ID NO: 199 or a sequence having at least 90%, atleast 95%, or at least 99% identity thereto. In some embodiments, theTCR beta variable domain comprises SEQ ID NO: 200 or a sequence havingat least 90%, at least 95%, or at least 99% identity thereto.

In some embodiments, the first or second ligand binding domain comprisesa sequence of any one of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214,SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222 Or SEQ IDNO: 224, or a sequence having at least 90%, at least 95% or at least 99%identity thereto.

It will be appreciated by the person of ordinary skill that first,activator ligand binding domains for the first receptor may be isolatedor derived from any source known in the art, including, but not limitedto, art recognized T cell receptors, chimeric antigen receptors andantibody binding domains.

Methods of Treatment

The present disclosure provides methods of treating a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of the engineered immune cells of the presentdisclosure.

The engineered immune cells of the present disclosure may be used totreat a subject that has cancer. The cancer may comprise a liquid tumoror a solid tumor. Exemplary liquid tumors include leukemias andlymphomas. Further cancers that are liquid tumors can be those thatoccur, for example, in blood, bone marrow, and lymph nodes, and caninclude, for example, leukemia, myeloid leukemia, lymphocytic leukemia,lymphoma, Hodgkin's lymphoma, melanoma, and multiple myeloma. Leukemiasinclude, for example, acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CIVIL), and hairy cell leukemia. Exemplary solid tumorsinclude sarcomas and carcinomas. Cancers can arise in virtually an organin the body, including blood, bone marrow, lung, breast, colon, bone,central nervous system, pancreas, prostate and ovary. Further cancersthat are solid tumors include, for example, prostate cancer, testicularcancer, breast cancer, brain cancer, pancreatic cancer, colon cancer,thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi'ssarcoma, skin cancer, squamous cell skin cancer, renal cancer, head andneck cancers, throat cancer, squamous carcinomas that form on the moistmucosal linings of the nose, mouth, throat, bladder cancer,osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer,liver cancer, and kidney cancer. In some embodiments, the conditiontreated by the methods described herein is metastasis of melanoma cells,prostate cancer cells, testicular cancer cells, breast cancer cells,brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroidcancer cells, stomach cancer cells, lung cancer cells, ovarian cancercells, Kaposi's sarcoma cells, skin cancer cells, renal cancer cells,head or neck cancer cells, throat cancer cells, squamous carcinomacells, bladder cancer cells, osteosarcoma cells, cervical cancer cells,endometrial cancer cells, esophageal cancer cells, liver cancer cells,or kidney cancer cells.

Treating cancer with the engineered immune cells of the presentdisclosure can result in a reduction in size of a tumor. A reduction insize of a tumor may also be referred to as “tumor regression”.Preferably, after treatment, tumor size is reduced by 5% or greaterrelative to its size prior to treatment; more preferably, tumor size isreduced by 10% or greater; more preferably, reduced by 20% or greater;more preferably, reduced by 30% or greater; more preferably, reduced by40% or greater; even more preferably, reduced by 50% or greater; andmost preferably, reduced by greater than 75% or greater. Size of a tumormay be measured by any reproducible means of measurement. The size of atumor may be measured as a diameter of the tumor.

Treating cancer with the engineered immune cells of the presentdisclosure can result in a reduction in tumor volume. Preferably, aftertreatment, tumor volume is reduced by 5% or greater relative to its sizeprior to treatment; more preferably, tumor volume is reduced by 10% orgreater; more preferably, reduced by 20% or greater; more preferably,reduced by 30% or greater; more preferably, reduced by 40% or greater;even more preferably, reduced by 50% or greater; and most preferably,reduced by greater than 75% or greater. Tumor volume may be measured byany reproducible means of measurement.

Treating cancer using the engineered immune cells of the presentdisclosure may result in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer with the engineered immune cells of the presentdisclosure can result in a decrease in number of metastatic lesions inother tissues or organs distant from the primary tumor site. Preferably,after treatment, the number of metastatic lesions is reduced by 5% orgreater relative to number prior to treatment; more preferably, thenumber of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer with the engineered immune cells of the presentdisclosure can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer with the engineered immune cells can result in anincrease in average survival time of a population of treated subjects incomparison to a population of untreated subjects. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer with the engineered immune cells can result in increasein average survival time of a population of treated subjects incomparison to a population receiving monotherapy with a drug that is nota compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer with the engineered immune cells can result in adecrease in the mortality rate of a population of treated subjects incomparison to a population receiving carrier alone. Treating cancer canresult in a decrease in the mortality rate of a population of treatedsubjects in comparison to an untreated population. Treating cancer canresult in a decrease in the mortality rate of a population of treatedsubjects in comparison to a population receiving monotherapy with a drugthat is not a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof.Preferably, the mortality rate is decreased by more than 2%; morepreferably, by more than 5%; more preferably, by more than 10%; and mostpreferably, by more than 25%. A decrease in the mortality rate of apopulation of treated subjects may be measured by any reproduciblemeans. A decrease in the mortality rate of a population may be measured,for example, by calculating for a population the average number ofdisease-related deaths per unit time following initiation of treatmentwith an active compound. A decrease in the mortality rate of apopulation may also be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing completion of a first round of treatment with an activecompound.

Treating cancer with the engineered immune cells can result in adecrease in tumor growth rate. Preferably, after treatment, tumor growthrate is reduced by at least 5% relative to number prior to treatment;more preferably, tumor growth rate is reduced by at least 10%; morepreferably, reduced by at least 20%; more preferably, reduced by atleast 30%; more preferably, reduced by at least 40%; more preferably,reduced by at least 50%; even more preferably, reduced by at least 50%;and most preferably, reduced by at least 75%. Tumor growth rate may bemeasured by any reproducible means of measurement. Tumor growth rate canbe measured according to a change in tumor diameter per unit time.

Treating cancer with the engineered immune cells can result in adecrease in tumor regrowth. Preferably, after treatment, tumor regrowthis less than 5%; more preferably, tumor regrowth is less than 10%; morepreferably, less than 20%; more preferably, less than 30%; morepreferably, less than 40%; more preferably, less than 50%; even morepreferably, less than 50%; and most preferably, less than 75%. Tumorregrowth may be measured by any reproducible means of measurement. Tumorregrowth is measured, for example, by measuring an increase in thediameter of a tumor after a prior tumor shrinkage that followedtreatment. A decrease in tumor regrowth is indicated by failure oftumors to reoccur after treatment has stopped.

Treating or preventing a cell proliferative disorder with the engineeredimmune cells can result in a reduction in the rate of cellularproliferation. Preferably, after treatment, the rate of cellularproliferation is reduced by at least 5%; more preferably, by at least10%; more preferably, by at least 20%; more preferably, by at least 30%;more preferably, by at least 40%; more preferably, by at least 50%; evenmore preferably, by at least 50%; and most preferably, by at least 75%.The rate of cellular proliferation may be measured by any reproduciblemeans of measurement. The rate of cellular proliferation is measured,for example, by measuring the number of dividing cells in a tissuesample per unit time.

Treating or preventing a cell proliferative disorder with the engineeredimmune cells can result in a reduction in the proportion ofproliferating cells. Preferably, after treatment, the proportion ofproliferating cells is reduced by at least 5%; more preferably, by atleast 10%; more preferably, by at least 20%; more preferably, by atleast 30%; more preferably, by at least 40%; more preferably, by atleast 50%; even more preferably, by at least 50%; and most preferably,by at least 75%. The proportion of proliferating cells may be measuredby any reproducible means of measurement. Preferably, the proportion ofproliferating cells is measured, for example, by quantifying the numberof dividing cells relative to the number of nondividing cells in atissue sample. The proportion of proliferating cells can be equivalentto the mitotic index.

Treating or preventing a cell proliferative disorder with the engineeredimmune cells can result in a decrease in size of an area or zone ofcellular proliferation. Preferably, after treatment, size of an area orzone of cellular proliferation is reduced by at least 5% relative to itssize prior to treatment; more preferably, reduced by at least 10%; morepreferably, reduced by at least 20%; more preferably, reduced by atleast 30%; more preferably, reduced by at least 40%; more preferably,reduced by at least 50%; even more preferably, reduced by at least 50%;and most preferably, reduced by at least 75%. Size of an area or zone ofcellular proliferation may be measured by any reproducible means ofmeasurement. The size of an area or zone of cellular proliferation maybe measured as a diameter or width of an area or zone of cellularproliferation.

Treating or preventing a cell proliferative disorder with the engineeredimmune cells can result in a decrease in the number or proportion ofcells having an abnormal appearance or morphology. Preferably, aftertreatment, the number of cells having an abnormal morphology is reducedby at least 5% relative to its size prior to treatment; more preferably,reduced by at least 10%; more preferably, reduced by at least 20%; morepreferably, reduced by at least 30%; more preferably, reduced by atleast 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. An abnormal cellular appearance or morphology may be measuredby any reproducible means of measurement. An abnormal cellularmorphology can be measured by microscopy, e.g., using an inverted tissueculture microscope. An abnormal cellular morphology can take the form ofnuclear pleiomorphism.

Exemplary methods of treatment and conditions to be treated using thecells of the present invention including those that have been disclosedby the present Inventors in PCT International Application Nos.PCT/US2019/037038, PCT/US2020/045250, PCT/US2020/045228,PCT/US2020/045373, and PCT/CA2016/051421, and U.S. ProvisionalApplication Nos. 62/946,888, 62/934,419, 63/076,123, 63/068,244,63/068,249, 63/068,245, 63/068,246, 63/065,324, and 63/037,975, whichare each incorporated herein by reference.

EXAMPLES Example 1

FIG. 4 provides experimental results showing that, for the engineeredimmune cells of the present disclosure expressing activating andblocking receptors, surface levels of the activating receptor decreasewhen the immune cells are in the presence of non-target cells expressingboth the activating and blocking ligand.

FIGS. 5-7 provides experimental results showing that this reducedsurface expression of the activating receptors corresponds with theability of the immune cells to kill other cells. Thus, advantageously,when the immune cells are in limited or no contact with target cells,their ability to kill is diminished, thereby reducing non-targeteffects.

Example 2

Another surprising facet of the immune cells of the present invention isthat the reduced surface expression of the activating receptors onlyoccurs when the immune cells contact non-target cells expressing boththe activating and blocking ligands. This is shown in FIG. 7.

Example 3

FIGS. 8-16 provide an experimental protocol and results that indicatethe reduced expression of activating receptors is reversible uponcontact with target cells.

Example 4

FIGS. 17-19 provide experimental results showing that, unlike theactivating receptor, the blocking receptor does not undergo reducedsurface expression in an appreciable amount in the presence ofnon-target cells.

Example 5

FIG. 22 shows experimental results indicating that the blocking receptorprovides a blocking signal that dominates and inhibits the activatingsignal from the activating receptor.

Jurkat cells were transfected with either an activating receptor(MP1-CAR) for a MAGE-A3 activating ligand or the activating receptor anda blocking receptor (ESO-Tmod) for a NY-ESO-1 blocking ligand.

Panel A shows the NFAT-luciferase signal of Jurkat cells transfectedwith either the activator alone or in combination with the blocker,after 6 hours of co-culture with activator and blocker peptide-loaded T2cells. The T2 cells were loaded with titrated amounts of activatorMAGE-A3 peptide and a fixed amount of blocker NY-ESO-1 peptideconcentration. This reveals the activation dose-response of thetransfected cells.

Panel B shows the NFAT-luciferase signal of Jurkat cells transfectedwith either the activator alone or in combination with the blocker,after 6 hours of co-culture with activator and blocker peptide-loaded T2cells. The T2 cells were loaded with titrated amounts of blockerNY-ESO-1 peptide and a fixed amount of activator MAGE-A3 peptideconcentration above the Emax concentration (˜0.1 μM). This reveals theinhibition dose-response of the transfected cells.

Panel C shows the NFAT-luciferase signal of Jurkat cells transfectedwith either the activator alone or in combination with the blocker,after 6 hours of co-culture with activator and blocker peptide-loaded T2cells. The x-value blocker NY-ESO-1 peptide concentrations from panel Bwere normalized to the constant activator MAGE peptide concentrationsused for each curve and plotted on the x-axis. The ratio of blockerpeptide to activator peptide required for 50% blocking (IC50) areindicated for each curve. The B:A peptide ratio required is less than 1indicating that, for this pair of activator CAR and blocker, similar (orfewer) blocker pMHC antigens may be sufficient on target cells to blockactivator pMHC antigens.

Since this ratio is less than 1, it can be inferred that the blockingsignal dominates and inhibits the activating signal. Thus, a singleblocking receptor can provide a blocking signal of sufficient strengthto inhibit the activating signal of one or more activating receptors. Assuch, the quantity of activating and blocking ligands expressed by anon-target cell can form part of the basis for determining theappropriate relative amounts of activating and blocking receptors thatshould be expressed by an immune cell of the disclosure.

Example 6

FIG. 23 provides experimental results showing that the blockingreceptors are ligand-dependent. For both CAR and TCR activatingreceptors, blocking receptors had minimal ligand-independent blockingactivity. This impact is shown by the minimal effect on the EC₅₀ of theactivating receptors by the blocking receptor in the presence/absence ofthe blocking ligand.

Example 7

FIG. 25 provides experimental results showing the relative impact hingelength and flexibility has on the strength of a blocking receptor as afunction of the EC₅₀ of the activating receptor.

Example 8

FIG. 26 shows the large relative impact of the LBD on the activatingreceptor's structure activity relationship when compared with theeffects provided by different hinges, transmembrane and intracellulardomains. In this study, 45 separate activating receptors were createdusing various combinations of ligand binding domains, hinges, andintracellular domains. For each receptor one of five ligand bindingdomains that bind to the same activating ligand were selected. Despiteall binding to the same target ligand, the identity of the ligandbinding domain caused differences in the EC₅₀ of the activatingreceptors that spanned orders of magnitude. The ligand binding domainwas shown to have greater than 10× the impact on the receptors' EC₅₀compared to the hinge, transmembrane and intracellular domains.

Example 9

FIGS. 33-34 show the impact receptor cross-talk can have on the abilityof the blocking receptor to inhibit the activation signal. Engineeredimmune cells were created with one of five different activatingreceptors. Though the activating receptors differed between the cells,each targeted the same activating ligand, epidermal growth factorreceptor (EGFR), using a different LBD. As shown by five graph panelsFIGS. 33-34, each of the different activating receptors provided theimmune cells with equivalent abilities to kill target cells. Then,immune cells were created that had one of the five activating receptorsand the same blocking receptor. Addition of the blocker caused some ofthe immune cells, like CT486-containing cells, to decrease their abilityto kill target cells.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

1. An engineered immune cell comprising: an activating receptorexpressed on a surface of the engineered immune cell, wherein binding ofthe activating receptor to a first ligand on a target cell promotes acytotoxic response by the engineered immune cell; and a blockingreceptor expressed on the surface of the engineered immune cell, whereinbinding of the blocking receptor to a second ligand on the target cellcauses the engineered immune cell to exhibit reduced surface expressionof the activating receptor.
 2. The engineered immune cell of claim 1,wherein binding of the blocking receptor to the second ligand on thetarget cell causes the blocking receptor to trigger an inhibitory signalthat blocks the activating signal, thereby preventing the cytotoxicresponse by the immune cell.
 3. The engineered immune cell of claim 2,wherein the inhibitory signal dominates and blocks the activatingsignal.
 4. The engineered immune cell of claim 1, wherein the reducedsurface expression of the activating receptor is reversible.
 5. Theengineered immune cell of claim 4, wherein the reduced surfaceexpression of the activating receptor reverses upon the engineeredimmune cell binding to the first ligand on a target cell in the absenceof the second ligand.
 6. The engineered immune cell of claim 1, whereinthe reduced surface expression of the activating receptor is localizedto a region of the engineered immune cell surface proximal to theblocking receptor.
 7. The engineered immune cell of claim 6, wherein aplurality of the blocking receptor binds to a plurality of the secondligand, and the reduced surface expression is localized to regions ofthe engineered immune cell surface proximal to blocking receptors. 8.The engineered immune cell of claim 1, wherein when the immune cellencounters a target cell having both the first and second ligands, aplurality of activating and blocking receptors diffuse into a region onthe of the immune cell surface proximal to the target cell and form amicro-cluster in which binding of blocking receptors to the secondligands causes the engineered immune cell to exhibit reduced surfaceexpression of the activating receptor.
 9. The engineered immune cell ofclaim 1, wherein the blocking receptor cannot bind to the second liganduntil the activating receptor binds to the first ligand.
 10. A methodfor treating a cancer, the method comprising: providing an engineeredimmune cell to a patient, wherein the engineered immune cell comprisesan activating receptor and a blocking receptor, each expressed on asurface of the engineered immune cell, wherein: when the engineeredimmune cell encounters a tumor cell of the patient, the activatingreceptor binds to a first ligand on the tumor cell while the blockingreceptor remains unbound, thereby promoting a cytotoxic response by theengineered immune cell that results in a cytotoxic effect on the tumorcell; and when the engineered immune cell encounters a normal cell ofthe patient the blocking receptor binds to a second ligand on the normalcell and causes the engineered immune cell to exhibit reduced surfaceexpression of the activating receptor, thereby causing a signal from theblocking receptor to dominate a signal from the activating receptor andprevent the cytotoxic response by the engineered immune cell.
 11. Themethod of claim 10, wherein the reduced surface expression of theactivating receptor is temporary.
 12. The method of claim 10, whereinthe reduced surface expression of the activating receptor is reversible.13. The method of claim 12, wherein the reduced surface expression ofthe activating receptor reverses upon the engineered immune cell bindingto the first ligand on a tumor cell.
 14. The method of claim 10, whereinthe reduced surface expression of the activating receptor is localizedto a region of the engineered immune cell surface proximal to theblocking receptor bound to the second ligand on the normal cell.
 15. Themethod of claim 14, wherein a plurality of the blocking receptor bindsto a plurality of the second ligand on the normal cell, and the reducedsurface expression is localized to the region of the engineered immunecell surface proximal to the plurality of the blocking receptor.
 16. Themethod of claim 10, wherein when the immune cell encounters at least onetumor cell, a plurality of the activating receptors diffuse into a firstregion on the surface of the immune cell proximal to the tumor cell andforms a first micro-cluster that promotes the cytotoxic response by theengineered immune cell that results in a cytotoxic effect on the tumorcell.
 17. The method of claim 16, wherein when the immune cellsimultaneously encounters a normal cell, a plurality of the activatingand blocking receptors diffuses into a second region on the surface ofthe immune cell proximal to the normal cell and forms a secondmicro-cluster in which the engineered immune cell exhibits reducedsurface expression of the activating receptor.
 18. The method of claim17, wherein the signal from the blocking receptors dominates the signalfrom the activating receptors in the second micro-cluster preventing thecytotoxic response by the immune cell on the normal cell.
 19. The methodof claim 17, wherein binding of the blocking receptors in the secondmicro-cluster to the second ligands prevents breakup of the secondmicro-cluster.
 20. The method of claim 17, wherein binding of theactivating receptors of the second micro-cluster to the first ligands ona tumor cell reverses the reduced surface expression of the activatingreceptor in the second micro-cluster.